Article pubs.acs.org/Langmuir
High-Resolution Ultrasonic Spectroscopy Study of Interactions between Hyaluronan and Cationic Surfactants Andrea Kargerová* and Miloslav Pekař Brno University of Technology, Faculty of Chemistry, Materials Research Centre, Purkyňova 118, 612 00 Brno, Czech Republic S Supporting Information *
ABSTRACT: Interactions in a cationic surfactant−hyaluronan system in water and in sodium chloride solution were investigated by high-resolution ultrasonic spectroscopy at 25 °C. Two alkyltrimethylammonium bromide surfactants of different chain lengths (tetradecyl and hexadecyl) were used; hyaluronan molecular weight ranged from 10 to 1750 kDa. Two main parametersultrasonic velocity and attenuationwere measured in the titration regime. Up to six different regions could be identified in the velocity titration profiles in water in a narrow interval of surfactant concentration. These regions differed primarily in the compressibility of structures formed in the system. The number of detected transitions was higher for the tetradecyl surfactant; therefore, the increased length of the hydrophobic chain simplified the details of the structure-forming behavior. The measurement of attenuation was much less sensitive and detected only the formation of microheterogeneous structures or visible phase separates. The richness of the titration profiles was depressed in salt solution, where essentially only two principal regions were observed. On the other hand, the effect of hyaluronan molecular weight on the positions of boundaries between regions was more significant in the presence of salt. Besides electrostatic interactions, hydrophobic interactions are also relevant for determining the behavior of hyaluronan−surfactant systems and the properties of formed complexes (aggregates).
1. INTRODUCTION Hyaluronan is a linear polysaccharide built from regularly alternating monosaccharides, glucuronic acid and N-acetylglucosamine, which form a basic disaccharide unit.1 Because of the presence of a dissociable (carboxyl) group on its basic unit, hyaluronan has the character of a polyelectrolyte. At physiological pH, the carboxyl groups are predominantly ionized, and hyaluronan behaves like a polyanion and can interact or associate with cationic counterions to maintain charge neutrality. Hyaluronan interactions with positively charged surfactants were thus studied as a specific case of polyelectrolyte−surfactant interaction.2,3 A series of papers by Swedish research groups provided a detailed study on the phase behavior of systems containing water, hyaluronan, alkyltrimethylammonium bromides (tetradecyl derivative was the most studied type), and salt (mostly NaBr).4−10 The binding of surfactant to hyaluronan was detected for surfactants with alkyl chains consisting of at least ten carbon atoms. The binding was found to be considerably weaker than for most other carboxylcontaining polyelectrolytes due to the low linear charge density of hyaluronan. Phase separation was observed as the formation of a precipitate or a gel-like phase upon increasing surfactant concentration. The phase diagrams constructed for the studied systems essentially contain an area of homogeneous single phase surrounding the two-phase region located around the water-rich corner of the phase triangle. The longer the hydrocarbon chain of the surfactant, the larger was the two© 2014 American Chemical Society
phase region. In contrast, hyaluronan molecular weight had only a slight effectit only slightly changed the position of the two-phase region while its size was almost unaffected. The results indicated the strong cooperativity of surfactant binding on the hyaluronan macromolecule, resulting in binding in the form of micelle-like clusters in which both hyaluronan carboxylates and background electrolyte anions participate as counterions. In order to be able to analyze the composition of separated phases, experiments on phase diagrams were conducted by mixing a hyaluronan solution (at a concentration usually around 1 wt %) with surfactant solutions of varying concentration and leaving the system to phase separate and then equilibrate for several days or weeks. This “static” experimental protocol is necessary when the individual equilibrated phases need to be characterized. Another approach is focused more on the study of interacting molecules or macromolecules shortly after coming into sufficiently close contact and gives a more dynamic picture of the system. Such experiments can be performed, for example, by means of conductometric11,12 or calorimetric13,14 titrations. In this work, high-resolution ultrasound spectrometry in titration regime is used. High-resolution ultrasound spectrometry is a powerful Received: May 13, 2014 Revised: August 6, 2014 Published: September 23, 2014 11866
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from a PURELAB (Option R7/15; ELGA, Great Britain) water purification system was used for the preparation of all samples. 2.2. Methods. Ultrasonic velocity and attenuation were measured at seven selected frequencies in the range from 2.5 to 17.5 MHz using an HR-US 102T ultrasonic spectrometer (Ultrasonic Scientific, Ireland) in the titration regime. This device is equipped with two cells enabling single-cell or differential measurements. The differential regime used in this work allows a resolution of 0.2 mm s−1 for ultrasonic velocity (this corresponds to 10−5% in water) and 0.2% for ultrasonic attenuation. The temperature was controlled with a Haake PC300 heating bath, which provided a temperature stability of ±0.01 °C. The temperature was recorded by a sensor imbedded in the body of the differential ultrasonic cell, as provided by the manufacturer. The measuring cell was filled with sample solution using a calibrated 1 mL Hamilton syringe. The exact amount of solution in the cell was determined by weighing the syringe before and after filling. The sample ultrasonic cell was closed with a stopper, which incorporated a line for the injection of titrant. The titration attachment to the HR-US 102T instrument was equipped with a 50 μL Hamilton syringe used to inject the titrant. One milliliter of deeply degassed sample was pipetted into the sample cell equilibrated at the selected temperature; 1 mL of deionized water had previously been pipetted into the reference cell. Ultrasonic measurements at the preselected frequencies commenced after the sample was loaded and temperature-equilibrated. Solutions of TTAB and CTAB in water or in 0.15 M NaCl were used as the titrant. The titrant solution was injected into the sample automatically in 2 μL steps by the computer-controlled titration attachment to the ultrasound spectrometer. The addition rate was controlled to avoid large kinetic effects (see also section 3.2.3) and to achieve stable data readings; preparatory measurements showed that the rate of one addition per 10 min is sufficient to this purpose. After each injection, the sample was stirred using a double stirring system that provided effective mixing within seconds. Ultrasonic velocity and attenuation were monitored continuously over the course of the titration at 25 °C. Before every measurement, baseline correction was done. The same measurements in the titration regime were performed for a sample cell loaded at time zero with deionized water or with 0.15 M NaCl. Ultrasonic velocity and attenuation profiles measured in water or in 0.15 M NaCl were subtracted from the corresponding profiles in samples in order to remove the effect of temperature fluctuations and fine differences in the resonance of the cells on the measured values of ultrasonic parameters. The measured data were processed using Titration Analysis software (Ultrasonic Scientific, Ireland), which also includes a correction for dilution. Each measurement was made in triplicate (or more times), and the values were averaged. Relative standard deviations in velocities were less than 2%; in attenuation, up to 10− 15%; and in concentrations reported in tables, less than 5%. Visual observation of the titrated systems was performed outside the spectrometer in a test tube with an upper and bottom stirrer, using the same steps of the titration procedure performed in the spectrometer. The surfactant/hyaluronan charge ratio was calculated assuming one negative charge on each hyaluronan disaccharide unit; the surfactants (TTAB and CTAB) were assumed to carry one positive charge on their molecule. The concentration of hyaluronan disaccharide units (in mol/L) was calculated as a quotient of the hyaluronan concentration (in g/L) and disaccharide molecular weight (401.299 g/mol).
technique for the analysis of colloids based on precision measurements of the parameters of high-frequency (ultrasound) sound waves propagating through analyzed samples.15−18 This technique allows the direct probing of microstructural organization and intermolecular forces in a variety of colloidal systems. Buckin et al.19 used high-resolution ultrasound spectrometry for the study of interactions of DNA with cationic surfactants. Combining ultrasound data with measurements of density, they found a high value for the effect of surfactant binding to DNA on compressibility. This could not be explained by hydration changes only and was considered as an indicator of the formation of micelle-like aggregates of surfactants on the DNA surface with a highly compressible core of the aggregates. Hickey et al.20 published an analysis of the phase diagram and microstructural transitions in a microemulsion system containing phospholipid in which a titration arrangement with the detection of ultrasound parameters was used. They demonstrated that break points (points of abrupt changes in slope) on dependences of ultrasound velocity on the concentration of titrant are located on phase boundaries and can be used to construct a phase diagram. Knowledge of other physical parameters of the microemulsion system also enabled a quantitative characterization of various detected microphases or the state of water to be made. A similar study on a compositionally different microemulsion system was later published by the same group.21 Singh et al.22 investigated interactions between poly(N-vinyl-2-pyrrolidone) and sodium dodecyl sulfate using less sensitive ultrasound equipment operating at a single frequency.23 Ultrasound velocity as a function of the surfactant concentration exhibited several breaks, which were attributed to different molecular organizations in the systemfor instance, the formation of premicellar associates or their binding to the polymer chain. The authors also noted that ultrasound titration did not substantiate previous claims based on conductivity or viscosity data. In this work, high-resolution ultrasound spectrometry was used to gain a more detailed insight into the dynamics of interactions between hyaluronan and oppositely charged surfactants: to study the effect of hyaluronan molecular weight or the length of the surfactant hydrophobic chain both in water and in model physiological solution. This technique has not previously been used to investigate hyaluronan−surfactant systems, and there are only two reports19,22 on its application in studies of polyelectrolyte−surfactant interactions.
2. MATERIALS AND METHODS 2.1. Materials. Hyaluronan samples were obtained from Contipro Biotech (Czech Republic), where they are produced by extraction from cell walls of the bacteria Streptococcus zooepidemicus. The product types are named according to the range of average molecular weights within which the molecular weight of a particular product falls. Four product types of different ranges were used in this work and are listed in Table ST1 of the Supporting Information, together with the true molecular weights of all used samplesseveral samples of the same product type were used due to limitations on resources and stability. The surfactants hexadecyltrimethylammonium bromide (CTAB) of 99% purity and tetradecyltrimethylammonium bromide TTAB of 98% purity were purchased from Sigma-Aldrich (Czech Republic), and sodium chloride of 99.5% purity was from Lachner (Czech Republic). Hyaluronan solutions were prepared at a concentration of 1 g/L (w0) by dissolving hyaluronan powder in deionized water or in 0.15 M NaCl. The mixture was stirred for 48 h in a closed vessel at room temperature to ensure complete dissolution. Ultrapure deionized water
3. RESULTS AND DISCUSSION High-resolution ultrasound spectrometry gives, as its output, the difference in velocity or attenuation measured between the sample and reference cells, i.e., in our case, the difference between the value in the sample and the values in water or NaCl solution. Data are therefore reported in terms of velocity difference (attenuation difference). 3.1. Critical Micellar Concentrations in Water and 0.15 M NaCl. First, titrations with pure surfactants in water or NaCl 11867
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solutions were performed for the purposes of verification and comparison. The measured velocity profiles are shown in Figures SF1 and SF2 of the Supporting Information. The velocity increased with increasing surfactant concentration due to the increasing number of surfactant molecules with hydration shells composed of less compressible water molecules.18 The increase was linear, and the point of abrupt change in the slope determines the critical micellar concentration. The decreased slope above the critical micellar concentration is a combined result of the formation of micelles with a compressible core, the release of stiff hydration water from surfactant monomers, and the formation of new hydration shells around micelles. The critical micellar concentrations were determined from the intersection of two straight-line segments fitting the ultrasonic velocity versus concentration plots and are close to the values given in the references (cf. Table ST2 in the Supporting Information). 3.2. Binding of Surfactants on Hyaluronan in Water. 3.2.1. Titration of Hyaluronan Solution by TTAB. Figure 1
Table 1. TTAB Concentrations at the Upper Boundaries of the Different Parts Identified in the Velocity Titration Profile and the Corresponding Charge Ratios for Hyaluronan of Different Molecular Weight water
0.15 M NaCl
Mw (kDa)
part
cTTAB (mmol/L)
charge ratio
10−30
I II III IV V I II III IV V I II III IV V I
0.37 3.0 4.0 4.6 6.3 0.35 2.0 2.6 3.0 7.2 0.48 2.7 3.2 3.9 8.3 0.38
0.15 1.3 1.7 2.0 2.9 0.14 0.9 1.1 1.3 3.5 0.19 1.2 1.3 1.7 4.0 0.15
II III IV V
2.7 3.0 4.0 8.4
1.1 1.3 1.7 4.0
90−130
300−500
1500− 1750
part
cTTAB (mmol/L)
charge ratio
I II III
0.60 0.24 see text
I II III
0.91 0.37 see text
I II III
0.37 3.2 3.6
0.15 1.4 1.6
I
0.6
0.23
II III
4.9 5.50
2.2 2.5
part II. In practice, ultrasound does not “see” the addition of surfactant molecules to the system. In this part, the binding of surfactant on hyaluronan in the form of micelles should prevail. Because the surfactant concentration is still below its normal critical micellar concentration, the micellization process is induced by the presence of hyaluronan, apparently by electrostatic interactions between oppositely charged groups on hyaluronan and the surfactant. The surfactant concentration corresponding to the beginning of part II can be thus considered as the critical aggregation concentration. Two opposite effects contribute to the almost constant ultrasound velocity in the second part. The formation of compressible micelles together with the release of the less compressible molecules of hydration water from the interaction sites decrease the velocity; the formation of new and rigid hydration shells on emerged micelles increases the velocity. The former effect thus more than compensates for the latter. Visually, the samples in part II changed from opalescent (at lower TTAB concentrations) up to milky clouded (at higher TTAB concentrations). The third part is characterized by a reincrease in ultrasound velocity upon the addition of surfactant. This part is somewhat similar to the premicellar part of the titration profile in pure water (with a smaller slope). At the end of this part (around the local maximum of ultrasound velocity), the formation of macroscopic phase separated particles was observed visually. In this part, the binding of surfactant in the form of micelles is finished, free (unbound) surfactant molecules probably appear in the solution, electrostatic repulsion is suppressed, and phase separation progresses to the macroscopic level. Both the free surfactant molecules and rigid phase separated particles contribute to the velocity increase.
Figure 1. Velocity titration profiles for TTAB in water and in hyaluronan solution (300−500 kDa, w0 = 1 g/L; frequency 15 MHz; 25 °C). The curve for the titration in water was shifted to overlap the first points of both profiles.
compares the ultrasound velocity titration curves obtained during the TTAB titration into pure water or hyaluronan solution (product type 300−500 kDa is presented as a typical example). For the sake of easy visual comparison, the curve measured in the absence of hyaluronan (cf. Figure SF1) was shifted by adding a constant so that both curves start at the same point. Up to six parts can be found for the titration profile measured in hyaluronan solution; surfactant concentrations at their boundaries are given in Table 1. In the first part, the shape of the velocity profile is practically identical to the corresponding premicellar part of the titration profile obtained in water. Visually, this part corresponds to clear solution; the charge ratio is well below one (see Table 1). In this part, no effect of added hyaluronan is thus observed, which should be because of the prevailing presence of free surfactant molecules; some single surfactant molecules could be loosely bound to hyaluronan without changing their compressibility. In the second part, the increase in ultrasound velocity with surfactant concentration stops at the presence of hyaluronan. In fact, a very slight velocity decrease is observed. This part resembles the postmicellar part of the titration profile in pure water. The charge ratio approached one almost up to the end of 11868
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In part IV the velocity decreasesthis time more steeply than in part II. The system progressively clarifies with increasing concentration of surfactant. At the same time, the number of macroscopic separates decreases, and at the end of part IV, only a few particles can be observed. The decrease in velocity is thus attributable to the decreased size and number of phase separated particles which are more rigid than the liquid medium. Part IV ends when the surfactant concentration corresponds to its normal critical micellar concentration. The velocity increases again during part V with a slope approximating to the slope of the premicellar part of the titration profile in water. The increase goes on in part VI but with a much lower slope similar to that of the postmicellar part of the titration profile in water. Visual inspection revealed no special changes or behavior in either part, just a slightly opalescent appearance. Taking into account the surfactant concentration, the formation of standard micelles should be expected in both parts. In part V, the formed micelles probably interact with the previously formed hyaluronan−TTAB complexes, dissolve the macroscopically separated particles and form rather rigid structures apparently of a more rigid microgel type, which increase the ultrasound velocity. When the interactions are completed, part VI begins, in which new free micelles are mainly formed. The measurement of ultrasound attenuation was much less sensitive as expected. From the transition between parts I and II the attenuation increased continuously up to the transition between parts III and IV (Figure SF3 in Supporting Information). Then, it decreased steeply in part IV and remained practically constant thereafter. Increasing attenuation indicates increasing heterogeneity of the system and the formation of bigger particles scattering the ultrasound wave. The step decrease corresponds to the dissolution of such particles and clarification of the system. Constant attenuation is found where no substantial heterogeneity evolves, i.e., when only really microscopic phase separation or structures are found. In contrast to velocity, attenuation values were dependent on the ultrasound frequency, which is typical for systems containing microheterogeneities capable of relaxation upon ultrasound propagation.20,24 Depending on the mechanism of relaxation, ultrasound waves of different frequency may be scattered to different degrees. However, the curved shapes of the attenuation profiles and their locations on the concentration axis were not affected by the frequency, as is illustrated in the typical example in Supporting Information (Figure SF4). 3.2.2. Titration of Hyaluronan Solution by CTAB. Titration profiles measured with CTAB are obviously different from those measured with TTAB. Some parts are missing in the velocity profile (see Figure 2)on the basis of changes in the profile slope, including its sign, it is reasonable to assume that parts I and III are missing, and part V extends through a much shorter concentration interval. The titration curve for the hyaluronan solution is, from the first addition of surfactant, different from that obtained for pure water. In comparison to TTAB, CTAB interacts with hyaluronan at a much lower surfactant concentration; consequently, part I was not observed. Part II is characterized also by the evolution of turbidity and extends to concentrations well above the normal critical micellar concentration; however, it terminates also around the equivalent charge ratio (Table 2). Visible macroscopic phase separation occurred here only in part IV, and the steep velocity decrease in this part is mainly a result of particles settling down out of the detection volume. The fact
Figure 2. Velocity titration profiles for CTAB in water and in hyaluronan solution (300−500 kDa, w0 = 1 g/L; frequency 15 MHz; 25 °C). The curve for the titration in water was shifted to overlap the first points of both profiles.
Table 2. CTAB Concentrations at the Upper Boundaries of the Different Parts Identified in the Velocity Titration Profile and the Corresponding Charge Ratios for Hyaluronan of Different Molecular Weight water
0.15 M NaCl
Mw (kDa)
part
cCTAB (mmol/L)
10−30
II IV V II IV V II IV V II
2.3 2.8 5.9 2.5 2.8 4.8 2.8 3.2 4.1 2.7
0.94 1.2 2.5 1.0 1.2 2.0 1.2 1.3 1.7 1.1
IV V
3.0 5.4
1.3 2.3
90−130
300−500
1500− 1750
charge ratio
part
cCTAB (mmol/L)
II III
see text
II III
see text
charge ratio
II III
3.1 3.8
1.3 1.6
II
5.0
2.1
III
6.0
2.6
that part III is missing is probably a result of the stronger cooperativity of CTAB binding on hyaluronan; perhaps no free surfactant molecules are present in this case. The precipitated particles were progressively dissolved during part V, changing from precipitates through clotty structures or swelled flocs to an almost clear system in part VI, where also the formation of free standard micelles can be expected. It is also interesting that the velocity difference measured in the TTAB−hyaluronan system approached the values measured for the TTAB micellar system and that the values were very close for both systems starting at a TTAB concentration of about 8 mmol/L (note that this is not seen in Figures 1 or 2 due to the shifting of curves). In contrast, the velocity difference in the CTAB−hyaluronan system was always higher than in corresponding hyaluronan-free solutions. CTAB thus forms complexes with hyaluronan which are more rigid and less compressible than CTAB micelles, whereas the opposite is found for TTAB. The titration profile of ultrasound attenuation measured for CTAB (Figure SF5 in Supporting Information) is also different 11869
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and kinetics of hyaluronan−surfactant interactions were adequately recorded during the ultrasound titrations and that potential progressive changes in ultrasound parameters would be caused by the progressive separation (sedimentation) of aggregates formed during the titration. 3.3. Effect of NaCl. The titration profiles were significantly changed by the presence of NaCl at a concentration of 0.15 M (the physiological concentration). In the case of TTAB (see Figure 3), the shapes of the titration profiles in the absence and
from that measured for TTAB (Figure SF3 in Supporting Information). It increases continuously, but only slightly, up to the boundary between parts II and IV (note: part III is missing). Then, it increases sharply (in contrast to the system with TTAB) up to the boundary between parts V and VI. In part VI it continues to increase at first, but with a lower slope, and is then essentially constant. The sharp increase reflects the increased heterogeneity due to the phase separation. The relatively high final value of attenuation, compared to the TTAB system, shows a more heterogeneous structure capable of more intensive scattering of ultrasound waves, formed probably by rather rigid and translucent micro- or nanogel particles. The effect of ultrasound frequency on both the velocity and attenuation titration profiles is very similar to the effect described above for TTAB. The results of ultrasonic titrations clearly show differences in the interactions of TTAB and CTAB with hyaluronan in water. Taking into account the molecular structure of these surfactants, the differences should be caused by the slightly longer alkyl chain of CTAB. In other words, besides the electrostatic contribution, hydrophobic interactions also play an important role, especially in the case of CTAB, which has a longer hydrophobic tail. For example, a greater (longer) hydrophobic component supports interactions at very low surfactant concentrations. 3.2.3. Effect of Molecular Weight. The effect of hyaluronan molecular weight on the shape of all measured velocity titration profiles was small. The shapes were retained, though some differences were found in the values of surfactant concentration corresponding to the boundaries between neighboring parts (see Tables 1 and 2) and in the velocity values within the same parts of the profile. Visual observation found that the size of precipitates increased with the increasing molecular weight of hyaluronan. The differences were particularly evident in parts III−VI and can be ascribed to the effects of differences in the conformations of hyaluronan chains of different molecular weight.26 On average, the two preparations of lower molecular weight differed from the two preparations of higher molecular weightthe latter gave very similar profiles. The effect on attenuation values was strongerthe higher the molecular weight, the greater the difference in attenuation values. This should be the result of more intensive ultrasound scattering from the more complex coiled structures formed from chains of higher molecular weight. 3.2.4. Stopped Titrations. As noted in the Introduction, many previous studies, especially those on phase diagrams4 or fluorescence studies,26 were based on mixing all components at the same time and letting the system stand for at least 24 h. The measurement of one experimental point during ultrasonic titration took about 10 min. To test the potential time development of our systems, several checking stopped titrations were performed. The titrations were stopped at three preselected points of the titration profile, and ultrasound parameters were measured during the following 13 h (see the example in Figure SF6 of the Supporting Information for the hyaluronan−CTAB system). When the titration was stopped in part II or IV, no further change in the parameters was observed. After stopping the titration in part V, a slight decrease in the velocity was detected (around 6% over the 13 h), which was attributable to the slow sedimentation of microgel particles. Attenuation, which is much less sensitive, did not show any change. The stopped titrations confirmed that the dynamics
Figure 3. Velocity titration profiles for TTAB in 0.15 M NaCl and in hyaluronan solution (300−500 kDa, w0 = 1 g/L; frequency 15 MHz; 25 °C). The curve for the titration in NaCl was shifted to overlap the first points of both profiles.
presence of hyaluronan practically coincided at low surfactant concentrations (and show the linear dependence of ultrasound velocity on the surfactant concentration; part I in Figure 3). In contrast to the titrations in water, this coincidence survived up to the critical micellar concentration. Yet, even after the critical micellar concentration, the velocity increased in NaCl solution in the presence of hyaluronan, though with an apparently somewhat smaller slope (part II in Figure 3). This continuous increase is interrupted by a short interval of almost constant velocity (part III in Figure 3), which corresponds to the steep increase in ultrasound attenuation (Figure 4) and to the visual observation of several macroscopically phase separated particles dispersed in a milky system. The charge ratio at the start point of this increase was about 1.3 (Table 1). It is interesting that this short interval occurs at surfactant concentrations close to the critical micellar concentration in water. The system is clear before and cloudy or milky after this interval. The ultrasound attenuation of the TTAB−hyaluronan−NaCl system remains almost unchanged up to the start of the short region of constant velocity, where it increases sharply (Figure 4). This sharp increase continues beyond the region of constant velocity, and then the attenuation progressively levels off and thereafter increases only moderately but continuously. In fact, the start of this only moderate increase in attenuation corresponds to a mild change in slope in the velocity titration profile (within the part IV). The increased attenuation reflects the formation of heterogeneities−microaggregates of hyaluronan−surfactant complexes. The effect of ultrasound frequency is similar as described abovethe velocity values are unaffected in contrast to the values of attenuation. The shapes of the titration profiles for CTAB in the hyaluronan−NaCl system (Figure SF7 in Supporting Informa11870
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this should not directly indicate that no interactions between hyaluronan and surfactant were detected. Further, only below the critical micellar concentration (0.58 mmol/L TTAB, 0.071 mmol/L CTAB) did the shapes (slopes) of profiles measured in the presence and absence of hyaluronan coincide. The progressive formation of rigid, very small structures which do not scatter ultrasound waves should be expected. The velocity profile measured for the molecular weight of 110−130 kDa shows, instead of the short interval of almost constant velocity, ̈ a broader interval of “inflexing” behavior, during which the attenuation also markedly increases and opacity develops. For the highest molecular weight (1500−1750 kDa), the constant velocity interval and corresponding sharp increase in attenuation are shifted to higher surfactant concentrations (4−6 mmol/L). Again, frequency affects the values of attenuation (not the shapes of profiles). As reviewed by Cowman and Matsuoka,25 in the presence of 0.15 M NaCl, hyaluronan in the low molecular weight region adopts free-draining conformation, whereas in the high molecular weight region non-free-draining ball-like conformation emerges. Short hyaluronan chains also favor intermolecular interactions, whereas longer chains do not show this effect. There is very little information on the effect of hyaluronan molecular weight on its interactions with cationic surfactants. Thalberg et al.3 determined the critical NaBr concentration needed in order to prevent phase separation in systems containing TTAB or dodecyl- or decyltrimethylammonium bromide with hyaluronan of two molecular weights (60 and 3100 kDa). The higher molecular preparation had a larger twophase region; i.e., it required a higher salt concentration to prevent separation at the same surfactant concentration. In water, a reduced molecular weight of the polysaccharide resulted only in a slight change to the position of the twophase region. Our results in the case of very low molecular weight hyaluronan seem to correspond to conclusions of ref 10in the presence of salt, the surfactant cation may replace the polysaccharide counterion, forming very small and phase nonseparating structures of separate surfactant molecules bound to the biopolymer chain. An increased molecular weight of hyaluronan leads to the formation of larger and coiled balllike biopolymer structures with surfactant molecules bound preferably on their surface. The larger the structure, the larger its surface and the higher the amount of surfactant which is needed to induce (micro)phase separation.
Figure 4. Attenuation titration profiles for TTAB in 0.15 M NaCl and in hyaluronan solution (300−500 kDa, w0 = 1 g/L; frequency 15 MHz; 25 °C). The curve for the titration in NaCl was shifted to overlap the first points of both profiles.
tion) are much closer to those for TTAB than those observed in water. The attenuation reflects the changes in velocity (Figure SF8 in Supporting Information). The effect of ultrasound frequency is still the same as described above. Because of the higher velocity and increasing attenuation at low CTAB concentrations in the presence of hyaluronan, it cannot be excluded that the surfactant interacts with CTAB in NaCl solution even in its normal premicellar region forming some more rigid structures. It cannot be concluded that micellar structures bound to hyaluronan are formed at this stage, but taking into account the smaller slope of velocity increase during the normal postmicellar region (at the CTAB concentration between ca. 1 and 3 mmol/L) single surfactant or “minimicelle”27 binding should be preferred. The steep increase in attenuation reflects increasing heterogeneity in the system, which results in the separation of macroscopic particles (the flakes). The interval of almost constant attenuation corresponds to the redissolving of big particles by the excess of surfactant and is followed by the progressive formation of rigid (increasing velocity) microheterogeneous (increasing attenuation in a cloudy system) structures, probably of microgel type, as can supposed due to the increased slope of the velocity profile (from the CTAB concentration of 6−7 mmol/L; see Figure SF7 in Supporting Information). The addition of salt thus suppressed most of the differences observed in the velocity titration profiles between the two surfactants in water. This might be surprising because electrolyte should screen electrostatic interactions, and the importance of hydrophobic interactions mediated by surfactant alkyl chains should be expected to increase. We will return to this finding later after presenting the effect of hyaluronan molecular weight. In contrast with the titrations in water, the effect of hyaluronan molecular weight was much more obvious in the presence of NaCl for both surfactants (example in Figure SF9 in the Supporting Information). The velocity profile for the lowest molecular weight (10−30 kDa) is practically linear, the attenuation is constant, and the sample is clear throughout the whole range of applied surfactant concentrations. Because the velocity systematically increases in the presence of hyaluronan,
4. CONCLUSION Because of its high sensitivity, high-resolution ultrasonic spectroscopy in titration regime gave a much more in-depth and dynamic picture of interactions when compared to other methods. The behavior of a hyaluronan−surfactant system is more complex than simple one-phase/two-phase behavior with a sharp boundary, as could be inferred from the phase diagrams. Up to six different regions could be identified in water in a narrow interval of surfactant concentrations corresponding to different states of hyaluronan−surfactant complexes formed by the interactions. These regions differed primarily in the shape of the dependence of ultrasound velocity on surfactant concentration values, which are determined by the compressibility of colloidal structures formed in the system. The basic disaccharide unit and its interaction abilities are relevant for hyaluronan interactions with oppositely charged surfactants in water, whereas specific conformations or coil shapes of the biopolymer chain of various lengths are not so important. 11871
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The richness of the titration profiles was depressed in salt solution, where essentially only two principal regions were observed. On the other hand, the effect of hyaluronan molecular weight on the position of the boundaries between regions was more significant in the presence of salt. The length of the surfactant hydrophobic tail had a very important effect on titration profiles in water. Hydrophobic interactions are thus also relevant for determining the behavior of hyaluronan− surfactant systems and the properties of formed complexes (aggregates). The more collapsed conformations supposed to exist in the presence of electrolyte as compared to conformations in water prevented the access of surfactant molecules to polyelectrolyte interaction sites and consequently restricted (cooperative) interactions between surfactant alkyl chains. In other words, the effect of salt on hydrophobic interactions was mediated by sterical causes.
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ASSOCIATED CONTENT
S Supporting Information *
Determination of critical micelle concentrations; attenuation titration profiles; results of stopped titrations; effect of frequency and hyaluronan molecular weight on the titration profiles. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail
[email protected] (A.K.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by projects No. LD12068 and LO1211 from the Czech Ministry of Education. This work is part of activities of the COST action CM1101.
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REFERENCES
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dx.doi.org/10.1021/la501852a | Langmuir 2014, 30, 11866−11872
