Langmuir 1999, 15, 4217-4221
4217
Rheological Properties of Sodium Hyaluronate in Decyltrimethylammonium Bromide Aqueous Solutions1 Kazuhiro Fukada,* Emi Suzuki, and Tsutomu Seimiya Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Minamiohsawa 1-1, Hachiohji, Tokyo 192-0397, Japan Received September 8, 1998. In Final Form: December 10, 1998 The interaction between sodium hyaluronate (NaHy) and decyltrimethylammonium bromide (C10TAB) has been investigated by phase behavior, viscosity, and dynamic shear moduli studies. When the concentration of C10TAB, Cs, was between 0.03 and 0.35 M and the polymer concentration was above 0.05 g L-1, the system separated into two phases. For the isotropic solutions containing a large excess of surfactant (Cs g 0.35 M), the boundary between the dilute and semidilute regime was determined by using the zero-shear specific viscosity data. Further, the intrinsic viscosity and the dynamic shear moduli of NaHy in C10TAB solutions were compared with those in NaCl solutions. The viscosity results suggested that NaHy and C10TAB formed compact complexes in 0.35 M C10TAB to give a small intrinsic viscosity and the size of the complexes increased with the increase of C10TAB concentration. When the added salt concentration was 1.00 M and the polymer concentration was in semidilute regime, it was suggested from the data on dynamic shear moduli that the entanglement density of Hy chains in C10TAB was higher than that in NaCl.
Introduction
Experimental Section
Hyaluronan (Hy, hyaluronate, hyaluronic acid), which is found in the extracellular matrix of mammals, is a linear polysaccharide whose molecular structure consists of a repeating disaccharide unit, D-glucuronic acid and Nacetyl-D-glucosamine (see the inset of Figure 1). At neutral pH, it is dissolved in water and behaves as a weakly charged polyelectrolyte due to the carboxylate group on each disaccharide unit. Hyaluronate in aqueous salt solutions is a wormlike chain characterized by an intrinsic persistence length ranging from 45 to 90 Å,2-4 and the distance between two consecutive ionic sites on the polymer chain has been estimated to be about 12 Å.5 To increase the knowledge of the function and behavior of hyaluronan in biological tissue, the interactions between Hy and amphiphilic molecules, e.g., cationic surfactants, have been investigated.6-9 Due to the favorable electrostatic interaction between Hy and cationic surfactants, the surfactants bind to Hy and influence the conformation of polymer chain and possible tendencies toward association with its neighbors. In the present study, we focused our attention on the effect of addition of decyltrimethylammonium bromide on the rheological properties of Hy solution expecting to obtain relevant information about the conformational changes and interactions between polyions.
Materials. The sample of sodium hyaluronate (NaHy) was provided by Shiseido Basic Research Lab. (Yokohama, Japan). This sample was isolated from a strain of bacteria, Streptococcus zooepidemicus, that produces hyaluronic acid extracellularly and was highly purified according to the method of Akasaka et al.10 Molecular weight of the sample was identified to be 1.4 × 106 as determined by the intrinsic viscosity, [η], measurements in 0.2 M NaCl using an Ubbelohde capillary viscometer at 25 °C. The reported relationship between [η] and viscosity-average molecular weight, [η] ) 0.0228Mv0.816, was employed for the calculation of Mv.11 A guaranteed grade reagent of decyltrimethylammonium bromide (C10TAB, Tokyo Kasei, Inc.) was purchased and used after recrystallization from an acetone/ethanol mixture. All solutions were prepared gravimetrically using purified water with Mill-Q Lab. (Millipore Co.). Methods. We used the following viscometers and rheometers depending on the composition of solutions studied: an Ubbelohde capillary viscometer, a rotational cone-and-plate viscometer (Tokimec; Visconic ELD and Viscorder VDU-3B measuring system, three cones with the following geometry were used: a 2.8-cm diameter and a 3° angle, a 4.8-cm diameter and a 1° 34′ angle, and a 4.8-cm diameter and a 48′ angle; covering shear rate 1-750 s-1), a VE stress controlled rheoanalyzer (Vilastic Scientific; covering shear rate 0.01-100 s-1), and a CVO rheometer (Bohlin Instruments; a cone-and-plate geometry with a 4-cm diameter and a 4° angle; covering shear rate 0.01-20 s-1). When the polymer concentration was below 1 g L-1 and the added salt concentration was 0.01 M or more, the NaHy solutions behaved as Newtonian and we used the Ubbelohde capillary viscometer to measure the viscosity. For solutions with polymer concentration between 1 and 10 g L-1, the dependence of viscosity on shear rate was measured by using the rotational viscometer or the VE stress controlled rheoanalyzer. When the polymer concentration was 10 g L-1 or more, the CVO rheometer was employed. The dynamic shear moduli, G′ and G′′, were measured by using the VE stress controlled rheoanalyzer and the CVO rheometer for NaHy solutions with the polymer concentrations below 10 and above 10 g L-1, respectively. All measurements were performed at 25 °C.
* To whom correspondence should be addressed. E-mail:
[email protected]. (1) Presented at Polyelectrolytes ’98, Inuyama, Japan, May 31-June 3, 1998. (2) Cleland, R. L. Biopolymers 1984, 23, 647. (3) Ghosh, S.; Li, X.; Reed, C. E.; Reed, W. F. Biopolymers 1990, 30, 1101. (4) Fouissac, E.; Milas, M.; Rinaudo, M.; Borsali, R. Macromolecules 1992, 25, 5613. (5) Preston, B. N.; Snowden, J. M.; Houghton, K. T. Biopolymers 1972, 11, 1645. (6) Thalberg, K.; Lindman, B. J. Phys. Chem. 1989, 93, 1478. (7) Thalberg, K.; Lindman, B.; Karlstrom, G. J. Phys. Chem. 1990, 94, 4289. (8) Thalberg, K.; Lindman, B. Langmuir 1991, 7, 277. (9) de Fatima Santos, S.; Nome, F.; Zanette, D.; Reed, W. F. J. Colloid Interface Sci. 1994, 164, 260.
(10) Akasaka, H.; Seto, S.; Yanagi, M.; Fukushima, S.; Mitsui, T. J. Soc. Cosmet. Chem. Jpn. 1988, 22, 35. (11) Cleland, R. L.; Wang, J. L. Biopolymers 1970, 9, 799.
10.1021/la9811822 CCC: $18.00 © 1999 American Chemical Society Published on Web 03/19/1999
4218 Langmuir, Vol. 15, No. 12, 1999
Figure 1. Phase diagram for the system NaHy-C10TABwater at 25 °C. Open circles refer to the composition where the system was one phase, and the filled circles refer to the composition where phase separation was observed. The viscosity-average molecular weight of NaHy was 1.4 × 106. The critical micelle concentration of C10TAB in pure water was 0.068 M. The boundary between the dilute and semidilute region was determined from the specific viscosity data (see text). Inset of (a): molecular structure of a repeating disaccharide unit of sodium hyaluronate. (b) the magnification of (a).
Results and Discussion Phase Diagram. Figure 1 is the phase diagram constructed by the naked eye observation of the NaHyC10TAB-water three-component system at 25 °C. When the concentration of C10TAB was between 0.03 and 0.35 M and the polymer concentration was above ca. 0.05 g L-1, a separation into two phases, one low viscous and one concentrated in NaHy, occurred as has been reported.6-8 In the high surfactant concentration region (g0.35 M), “redissolution” of the precipitate occurred. It is believed that micellelike surfactant aggregates are bound to the Hy chains in the concentrated cationic surfactant solutions.6-9 Thalberg et al. have already reported the phase diagrams of aqueous systems of NaHy and cationic surfactants of the alkyltrimethylammonium bromide type with the number of carbon atoms from 8 to 16 (CnTAB; n ) 8-16). They found that binding of the surfactant (except for C8TAB and C9TAB) to Hy chains started at a well-defined concentration, denoted by c1, below the critical micelle concentration (cmc) of the surfactant and resulted in the phase separation.6 Also, they have found that by adding a large excess of the surfactant “redissolution” of the precipitate occurs in a same way as indicated in Figure 1. They had studied the NaHy-alkyltrimethylammonium
Fukada et al.
bromide-water systems with focus on the phase-separating samples and reported the mechanisms behind the redissolution phenomena based on the Flory-Huggins theory7 and the structure of the concentrated gel-like phase of the phase-separating samples by measuring 1H NMR transverse relaxation and self-diffusion.8 In the present study, on the other hand, we focus on the isotropic region where a large excess of surfactant coexists with NaHy from the viewpoint of rheological properties. Viscosity of NaHy-C10TAB and NaHy-NaCl Solutions. Figure 2 is an example of a double-logarithmic plot of the viscosity vs shear rate for NaHy-C10TAB and NaHy-NaCl solutions. It can be seen that when the polymer concentration, Cp, is relatively low, the viscosity is independent of shear rate. We confirmed that when Cp was below 1 g L-1 and the added salt concentration, Cs, was 0.01 M or more, the values of η obtained by the Ubbelohde capillary viscometer and by the other viscometers were the same within the experimental error. With the increase of Cp, however, non-Newtonian flow behavior became noticeable and the zero-shear rate viscosity, η0, was obtained from the Newtonian region of the flow curve. Then, the specific viscosity, ηsp0, was calculated according to the equation ηsp0 ) (η0 - η1)/η1, where η1 is viscosity of C10TAB or NaCl solutions. It is well-known that viscosity of polymer solutions is a valuable property of molecular size and interaction and that polymer concentration dependence of zero-shear viscosity shows different behaviors depending on the range of the polymer concentration. With the increase of Cp the slope of the double-logarithmic plot of ηsp0 vs Cp changes, and one can define the dilute and semidilute regime of polymer solutions. In a dilute region where polymer chains are isolated in solution, the reduced viscosity, ηsp0/Cp, is expressed in expansion form as
ηsp0/Cp ) [η] + k′ [η]2Cp + ...
(1)
where k′ is Huggins’ constant and [η] is the intrinsic viscosity. So far, viscosity of polyelectrolyte solutions has been studied mainly in the dilute regime, and their intrinsic viscosity was discussed in terms of expansion factors if the ionic strength is not lower than 0.01.12,13 In the semidilute region, on the other hand, polymer chains are entangled and the rheological properties are controlled by the degree of chain overlapping, and the concentration dependence of the specific viscosity can be expressed by scaling laws.13,14 Figure 3 shows double-logarithmic plots of ηsp0 vs Cp and linear plots of ηsp0/Cp vs Cp in aqueous solutions of C10TAB and NaCl for comparison. It is confirmed from parts a and c of Figure 3 that when Cp is larger than 3 g L-1 the polymer concentration dependence of ηsp0 in 0.35 and 1.00 M C10TAB and in 0.20, 0.35, and 1.00 M NaCl is approximately given by ηsp0 ∝ Cp4.25. Fouissac et al.15 have reported that the slopes of log ηsp0 vs log Cp in 0.1 M NaCl for NaHy samples with molecular weights in the range 3.5 × 105 to 2.2 × 106 were 4.0 ( 0.2 in the semidilute regime. So, we may safely say that our NaHy sample is in semidilute regime in the above-mentioned mediums when Cp is larger than 3 g L-1. At lower polymer concentrations, i.e., below 1 g L-1, the reduced viscosities (12) Noda, I.; Tsuge, T.; Nagasawa, M. J. Phys. Chem. 1970, 74, 710. (13) Yamaguchi, M.; Wakutsu, M.; Takahashi, Y.; Noda, I. Macromolecules 1992, 25, 470. (14) Takahashi, Y.; Isono, Y.; Noda, I.; Nagasawa, M. Macromolecules 1985, 18, 1002. (15) Fouissac, E.; Milas, M.; Rinaudo, M. Macromolecules 1993, 26, 6945.
Rheological Properties of NaHy in Surfactant Solutions
Langmuir, Vol. 15, No. 12, 1999 4219
Figure 2. Shear rate dependence of viscosity of NaHy solutions in various concentrations of C10TAB (a) and NaCl (b) at 25 °C. Cp (Cs) refers to NaHy (C10TAB or NaCl) concentration. Table 1. Intrinsic Viscosity, [η], and Huggins Constant, k′, in C10TAB and NaCl Solutions in C10TAB solutions C10TAB/M 0.03 0.35 0.50 1.00
[η]/L
g-1
3.96 0.96 1.38 1.80
in NaCl solutions k′
NaCl/M
[η]/L g-1
k′
0.35 2.02 1.00 0.95
0.01 0.03 0.20 0.35 1.00
5.33 3.76 1.83 1.92 1.34
0.40 0.48 0.62 0.75 0.90
were given by a linear function of Cp (see parts b and d of Figure 3) characterizing the dilute region where Huggins law (eq 1) applies. Further, the approximate relation ηsp0 ∝ Cp1.2 was found in the dilute regime as has been reported by Fuissac et al.,15 and the boundary between the dilute and semidilute regime,Cp*, was estimated from the intersection of the two slopes. The values of Cp* were 1.9 g L-1 in 0.35 M C10TAB and 2.4 g L-1 in 1.00 M C10TAB. Almost the same values for Cp* were obtained in 0.35 and 1.00 M NaCl, respectively. Although these results seemingly suggest that the molecular size and intermolecular interactions of Hy chains in the concentrated C10TAB are not very different from those of Hy chains in NaCl solutions with the corresponding concentration, one can see differences between the two mediums by the close inspection of Figure 3 as will be described hereinafter. From the intercepts and the slopes of curves in parts b and d of Figure 3, the values of [η] and k′ were obtained, and they are summarized in Table 1. We can see that in NaCl solutions monotonic decrease of [η] and increase of k′ with the added salt concentration is confirmed. In C10TAB solutions, on the other hand, [η] (k′) has minimum (maximum) at 0.35 M. By plotting [η] as a function of the inverse of square root of salt concentration, the different behavior of [η] in the two mediums is clearly demonstrated (see Figure 4). While the linear relationship between [η] and Cs-1/2 holds in 0.01-1.00 M NaCl indicating the gradual expansion of Hy chains with the decrease of ionic strength, [η] decreases with increasing Cs-1/2 in 0.35-
1.00 M C10TAB. At present, it is difficult to explain the reason for the different dependence of [η] on Cs-1/2 in C10TAB and NaCl in terms of ionic strength since the activity of ions in micellar solutions is very different from those in simple 1/1 electrolyte solutions even if the molarity is the same. However, a possible qualitative interpretation for the Cs dependence of [η] in C10TAB solutions may be as follows: Hy chains and C10TAB form some soluble complexes with compact structure in 0.35 M C10TAB, and the size of Hy-C10TAB complexes increases with the increase of C10TAB concentration to give larger [η]. Further studies estimating intrinsic viscosities of NaHy in aqueous solutions of C8TAB, which does not induce phase-separation since the interaction with Hy chains is weak, will aid in understanding the present results. Dynamic Shear Moduli of NaHy-C10TAB and NaHy-NaCl Solutions. We measured the dynamic shear moduli for the samples in a semidilute regime where the polymer chains are mutually entangled (see Figure 5). Parts a-c of Figure 5 show the storage and loss elastic moduli, G′ and G′′, respectively, at various frequencies for NaHy-NaCl (1.00 M) solutions with the polymer concentrations of 20, 10, and 4 g L-1, respectively. In parts a and b of Figure 5, there appears to be an intersection of the G′ and G′′ curves at 0.4 and 1.4 Hz, respectively, and G′ and G′′ above the intersection tend to level off and approach plateau values suggesting the existence of an entanglement network of Hy chains. When the polymer concentration is lowered to 4 g L-1 (Figure 5c), the intersection disappears from the frequency range studied. These results indicate the weakened network of Hy chains by the reduction in the entanglement density with decreasing polymer concentration. Yanaki and Yamaguchi16 have reported the dynamic shear moduli at various frequencies for NaHy samples with various molecular weights ranging from (0.13 to 2.15) × 106 in 0.147 M saline solutions (the polymer concentration was fixed at 1%) and (16) Yanaki, T.; Yamaguchi, T. Biopolymers 1990, 30, 415.
4220 Langmuir, Vol. 15, No. 12, 1999
Fukada et al.
Figure 3. Dependence of zero-shear specific viscosity, ηsp0, and the reduced viscosity,ηsp0/Cp, on NaHy concentration in aqueous solutions of C10TAB (a, b) and NaCl (c, d). Cp refers to NaHy concentration. The solid and broken lines in a and c indicate the slope ηsp0 ∝ Cp4.25 and ηsp0 ∝ Cp1.2, respectively.
Figure 4. Dependence of intrinsic viscosity, [η], on the inverse of square root of the concentration of C10TAB (b) or NaCl (O),Cs-1/2. The broken line indicates the reported intrinsic viscosity data for the NaHy sample with molecular weight 1.87 × 106 in various concentrations of NaCl aqueous solutions.4
demonstrated the reduction in the entanglement density of Hy chains with the decreasing molecular weight. It is interesting to compare parts c and d of Figure 5, in which the polymer concentration is the same but the added salt is NaCl (C10TAB) in Figure 5c (5d). One can see that while the loss modulus dominates at all frequencies for the NaHy-NaCl system, an intersection of the G′ and G′′ curves is observed at ca. 1.5 Hz and G′ exceeds G′′ at higher frequencies for the NaHy-C10TAB system, sug-
gesting the higher entanglement density of Hy chains in C10TAB than in NaCl. (Note that 1.00 M C10TAB solution without NaHy exhibits no elastic response.) Since the intrinsic viscosity of NaHy in 1.00 M C10TAB is larger than that in 1.00 M NaCl (see Table 1), one may consider the expanded structure of Hy-C10TAB complexes in 1.00 M C10TAB, and therefore the entanglement density in semidilute regime may become higher in 1.00 M C10TAB than in 1.00 M NaCl. To confirm the validity of this interpretation, scattering techniques should be applied to the NaHy-C10TAB system to get information on Hy chain dimensions. It is also to be noted here that Lindman and Thalberg17 have reported the dynamic shear moduli for the NaHy-C14TAB system in a gel-like region and no fundamental difference in elastic properties was found between NaHy solutions with and without surfactant, pointing to a loose and dynamic character of the binding of alkyltrimethylammonium-type surfactants to Hy chains. Conclusion Rheological properties of aqueous solutions of NaHy in an oppositely charged surfactant, C10TAB, and in a simple 1/1 electrolyte, NaCl, has been investigated. It is found that while [η] linearly increases with Cs-1/2 in 0.01-1.00 (17) Lindman, B.; Thalberg, K. Interactions of Surfactants with Polymers and Proteins; CRC Press: Boca Raton, FL, 1993; pp 259-261.
Rheological Properties of NaHy in Surfactant Solutions
Langmuir, Vol. 15, No. 12, 1999 4221
Figure 5. Storage and loss elastic moduli, G′ and G′′, respectively, as a function of oscillation frequency for NaHy solutions in 1.00 M NaCl (a, b, c) and in 1.00 M C10TAB (d) at 25 °C. The concentrations of NaHy are 20.0 g L-1 (a), 10.0 g L-1 (b), and 4.0 g L-1 (c, d), respectively.
M NaCl, [η] decreases with increasing Cs-1/2 in 0.35-1.00 M C10TAB. Also, the data of dynamic shear moduli for semidilute NaHy solutions suggests the higher entanglement density of Hy chains in 1.00 M C10TAB than in 1.00 M NaCl. A possible qualitative interpretation for these results may be as follows: Hy and C10TAB form some complexes with compact structure in 0.35 M C10TAB, and the size of the complexes increases with the increase of
C10TAB concentration to give higher [η] and higher entanglement density of Hy chains. Acknowledgment. We thank Ms. Atsuko Yamagishi, Jasco International Co., for the measurements of the dynamic shear moduli using the VE stress controlled rheoanalyzer and the CVO rheometer. LA9811822
