Effects of β-Cyclodextrin Addition and Temperature on the Modulation

Novel information about the effects of β-cyclodextrin (β-CD) addition and temperature on structural and rheological features of semidilute solutions...
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Biomacromolecules 2005, 6, 3129-3136

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Effects of β-Cyclodextrin Addition and Temperature on the Modulation of Hydrophobic Interactions in Aqueous Solutions of an Associative Alginate Anna-Lena Kjøniksen,† Ce´ line Galant,‡ Kenneth D. Knudsen,‡ Giao T. M. Nguyen,† and Bo Nystro¨m*,† Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, N-0315 Oslo, Norway, and Department of Physics, Institute for Energy Technology, P.O. Box 40, N-2027 Kjeller, Norway Received July 1, 2005; Revised Manuscript Received September 20, 2005

Novel information about the effects of β-cyclodextrin (β-CD) addition and temperature on structural and rheological features of semidilute solutions of alginate and its hydrophobically modified analogue (HMalginate) is given. Enhanced turbidity is observed for the HM-alginate solutions at high levels of β-CD addition and low temperatures. The viscosity results revealed cross-linking of the alginate chains at high β-CD concentrations and low temperatures. Rheological results for the HM-alginate solutions demonstrated that high levels of β-CD addition and elevated temperatures promoted decoupling of the hydrophobic polymer-polymer associations via inclusion complex formation between β-CD cavities and the hydrophobic side chains of the polymer. Analysis of small-angle neutron scattering (SANS) results from HM-alginate solutions in the presence of β-CD suggested that the polymer chains are locally stretched at all of the considered levels of β-CD and temperatures. The SANS results revealed association structures. The general picture that emerges is that β-CD addition and temperature can be combined to tune the intensity of the hydrophobic interactions and to cross-link the unmodified alginate. Introduction The interaction between nonionic hydrophobically modified water-soluble polymers and ionic surfactants in aqueous solution has been the subject of intense scrutiny in recent years.1-6 Some of this activity has been focused on structural and dynamical features in mixtures of amphiphilic polymer and surfactant. The binding of an ionic surfactant to a nonionic polymer endows a polyelectrolyte character to the polymer, and the physical behavior of the system is governed by a delicate interplay between hydrophilic, hydrophobic, and electrostatic interactions. In semidilute polymer solutions, this synergism between polymer and surfactant usually leads to the formation of association complexes and a viscosification of the mixture at low surfactant concentration, whereas at higher levels of surfactant addition the solubilization of the hydrophobic microdomains results in a disruption of the network. Thus, the strength of association can be tuned by changing the polymer-surfactant composition. However, this type of system is rather complex because of the electrostatic interactions induced by the ionic surfactant, and most commercial surfactants are toxic and therefore not suitable for, for example, pharmaceutical applications. Another mechanism to modulate the intensity of hydrophobic associations is to utilize the biocompatible cyclodextrin that forms inclusion complexes with polymers.7-11 * To whom correspondence should [email protected]. † University of Oslo. ‡ Institute for Energy Technology.

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Cyclodextrins (CDs) are cyclic starch oligomers consisting of 6, 7, or 8 (R-1,4)-linked R-D-glucopyranose units, and named R-, β-, and γ-CD, respectively.8 The apolar nature of their cavities8 allow CDs to act as hosts for both nonpolar and polar guests, which include small molecules as well as polymers. The crystal structures of cyclodextrin inclusion complexes can be classified into two main categories:12 “cage” and “channel” structures, where the channel-type crystalline structure is most common for long-chain molecules such as polymers.8 As a result of this process, pendent hydrophobic moieties of a polymer chain can be included inside these nanoscale channels of cyclodextrin, and thereby the tendency to form hydrophobic associations is reduced. This deactivation of the hydrophobic groups can be used to tune the hydrophobic interactions, and this type of siteselective complexation may play an important role in the construction of artificial supramolecular structures in polymeric systems. In a previous study,13 we reported the effect of β-CD addition to semidilute aqueous solutions of alginate and a hydrophobically modified analogue (HM-alginate) at a fixed temperature (25 °C). Alginate is an anionic copolymer comprised of residues of β-D-mannuronic acid and R-Lguluronic acid, and the polymer backbone of the hydrophobically modified analogue is equipped with pendent C8 chains, which can be encapsulated and deactivated through the β-CD cavities. It was demonstrated that an increasing amount of β-CD to the HM-alginate solution clearly reduced the hydrophobic associations and both the structural and rheological properties of the system were significantly

10.1021/bm050458z CCC: $30.25 © 2005 American Chemical Society Published on Web 10/06/2005

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altered. In the present work, novel information about the combined effect of β-CD concentration and temperature on the association behavior in semidilute aqueous solutions of alginate and HM-alginate will be presented. The surmise is that β-CDs form inclusion complexes through noncovalent interactions with the hydrophobic guest moieties, and since the water solubility of β-CD increases with increasing temperature, the intensity of the interaction between polymers and β-CD is expected to be changed. It will be demonstrated that temperature is a powerful variable to modulate the strength of the decoupling of the hydrophobic tails in the presence of β-CD. Several novel effects of temperature on the structural and rheological features will be reported. The objective of this investigation is to characterize the intricate interplay between the temperature effect and the level of added β-CD in alginate systems. For this purpose, we have carried out turbidity, small-angle neutron scattering (SANS), and rheological measurements on semidilute solutions of alginate and HM-alginate at different β-CD concentrations and temperatures. The corresponding experiments on the solutions of the unmodified alginate serve as reference in the process of evaluating the effect of the hydrophobic associations. The intention with the turbidity measurements is to keep track of significant changes of the thermodynamic conditions of the systems and to determine the cloud point (CP) under various conditions of β-CD addition and temperature. Structural changes of the systems on a mesoscopic dimensional scale are characterized by means of SANS, and the viscoelastic features of the systems are probed with rheological methods. Experimental Section Materials and Solution Preparation. An alginate sample, designated LF 10/60 LS, was supplied by FMC Biopolymers, Drammen, Norway. According to the specifications from the manufacturer, this sample has a weight-average molecular weight of 152 000, and the guluronic acid to mannuronic acid (G/M) ratio is 0.75. Dilute alginate solutions were dialyzed against pure water for several days to remove the salt and other low-molecular weight impurities. Regenerated cellulose with a molecular weight cutoff of 8000 was used as the dialyzing membrane. β-Cyclodextrin was supplied by Fluka and was used without further purification. The hydrophobically modified analogue, with attached C8 groups, was synthesized from the parent alginate sample by utilizing the coupling agent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC-HCl) to form amide linkages between amine containing molecules and the carboxylate moieties on the alginate polymer backbone. This method has been described elsewhere14-16 and the characteristic data of the hydrophobically modified sample has been reported recently.13 From quantitative 1H NMR spectroscopy and elemental analysis, the HM-alginate was found to contain 31 mol % of C8 groups. Polymer solutions with a fixed concentration of 2 wt % were prepared by weighing the components, and the samples were dissolved in heavy water, followed by stirring for a few days to allow the semidilute solutions to homogenize. In all of the experiments, heavy

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water was used as a solvent to obtain fully comparable results from the different experimental techniques (D2O yields a better contrast in the neutron scattering experiments). Turbidity Measurements. The turbidities of the β-CD/ D2O solutions and of the polymer/β-CD/D2O systems were determined with the aid of an NK60-CPA cloud point analyzer from Phase Technology, Richmond, B. C., Canada, and a temperature-controlled Helios Gamma (Thermo Spectronic, Cambridge, U.K.) spectrophotometer, respectively. The specifications of the cloud point analyzer and the determination of turbidities have been reported elsewhere.17 The measured signal S from the cloud point analyzer can empirically be related to the turbidity τ, which was determined from measurements of the transmittance on a standard spectrophotometer in a 1 cm cuvette, using the expression τ ) (-1/L) ln(It/I0), where L is the light path length of the cuvette, It is the transmitted light intensity, and I0 is the incident light intensity. A direct relationship between the determined turbidity from the spectrophotometer measurements and S from the cloud point analyzer is found17 to be τ ) 9.0 × 10-9 S3.751. Henceforth, all data from the cloud point analyzer will be presented in terms of turbidity. The very accurate temperature control of the sample in this instrument and the registration of the diffuse scattered light from the surface of the plate make this a powerful apparatus to monitor turbidity changes in connection with crystallization of β-CD in water. Conventional measurements of the transmitted light intensity through ordinary spectrophotometer cuvettes are usually unsuitable due to sedimentation of large clusters during the crystallization process. In the turbidity measurements of the viscous polymer/βCD mixtures, the formation of air bubbles onto the plate of the turbidimeter constituted a serious problem, and it was difficult to remove these bubbles from the viscous layer on the plate. In this case, no effect of sedimentation was observed, and the turbidities of the samples were determined with the Helios Gamma spectrophotometer at different temperatures at a wavelength of 500 nm. The air bubbles from the samples in the cells were removed by centrifuging the solutions at 3000 rpm for 15 min prior to measurement. The experiments were started at the highest studied temperature and they were carried out in 1 cm cuvettes with stoppers. At each temperature of measurement, the sample was allowed to equilibrate for 1 h before the commencement of the experiment. Even longer waiting times were tested, but no effect of time on the results was observed. SANS Measurements. Small-angle neutron scattering (SANS) experiments were carried out on 2 wt % solutions of HM-alginate in the presence of various amounts of β-CD and at different temperatures (temperature controlled to within (0.1 °C) at the SANS installation at the IFE reactor at Kjeller, Norway. The details of the spectrometer have been described elsewhere.17 The solutions were filled in 2 mm Hellma quartz cuvettes, which were placed onto a copperbase for good thermal contact and mounted in the sample chamber. The chamber was evacuated to reduce air scattering. The scattering length density calculated for the β-CD monomers (Fβ-CD ) 0.88 × 1010 cm-2) is quite close to that of HM-alginate (Fβ-CD ) 1.46 × 1010 cm-2). For this

Effects of β-CD on an Associative Alginate

reason, the contrast matching method18 has not been used to observe the scattering from the individual components within the mixtures. However, it should be mentioned that the scattering from β-CD in the samples is insignificant in comparison with that of the HM-alginate. Each complete scattering curve is composed of three independent series of measurement, using three different wavelength-distance combinations (5.1 Å/1.0 m, 5.1 Å/3.4 m, and 10.2 Å/3.4 m). By using these combinations, scattering vectors q ) (4π/λ) sin(θ/2) (where θ is the scattering angle) in the range of 0.008-0.25 Å-1 were covered. Standard reductions of the scattering data, including transmission corrections, were conducted by incorporating data collected from empty cell, beam without cell, and blocked-beam background to obtain the scattered intensities in absolute scale (cm-1). Subtraction of the solvent scattering was then done to obtain the coherent macroscopic scattering cross section dΣ/dΩ (q) of the system. Rheological Experiments. Shear viscosity and oscillatory sweep measurements were conducted with a Paar-Physica MCR 300 rheometer using a cone-and-plate geometry, with a cone angle of 1° and a diameter of 75 mm. The samples were introduced onto the plate, and to prevent evaporation of the solvent, the free surface of the sample was always covered with a thin layer of low-viscosity silicone oil (the viscoelastic response of the samples is not affected by this layer). The measuring device is equipped with a temperature unit (Peltier element) that provides a rapid alteration of the temperature and gives an effective temperature control over an extended time for all of the temperatures investigated in this work. The values of strain amplitude were checked to ensure that all measurements were performed in the linearviscoelastic regime, where the dynamic storage modulus (G′) and loss modulus (G′′) are independent of strain amplitude. The oscillating sweep experiments were carried out over an extended angular frequency (ω) domain. The shear viscosity measurements were conducted over an extended shear rate range. Results and Discussion Turbidimetry. β-CD is a torus-shaped molecule, with 15.4 Å outer diameter, 6 Å cavity diameter, 8 Å height, and a volume of approximately 1500 Å3.19 One rim of the torus is lined with O2 and O3 hydroxyl groups and the other with O6 hydroxyl groups; that is, β-CD is hydrophilic at the outside but the cavity contains C-H groups and is of hydrophobic character. During the crystallization process that occurs at low temperatures, CDs assume a herringbone-like arrangement where the cavity of one molecule is blocked on both sides by adjacent, symmetry-related β-CD molecules (cage arrangement) and the crystallite structure is stabilized by hydrogen bonds and van der Waals interactions. It has been reported20 from previous light microscopy studies that the β-CD crystallites, formed at low temperature, are thin parallelepipeds with an approximate axial ratio 1:2 when their size reaches a few micrometers. Figure 1 presents turbidity (τ) versus temperature curves for β-CD dissolved in D2O in the absence of polymer at

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Figure 1. Illustration of the temperature effect on the turbidity behavior for the β-CD/D2O system. The inset shows the effect of β-CD concentration on the cloud point, which is determined from the onset of the increase of the turbidity while lowering the temperature.

different concentrations during the crystallization process in down-scans at a slow cooling rate (0.05 °C/min). The characteristic feature is the enhanced turbidity by lowering the temperature, and this effect appears at higher temperature and becomes more pronounced as the concentration of β-CD is increased. This behavior announces aggregation upon cooling and it reflects the poorer water solubility of β-CD at lower temperatures. The augmented turbidity observed at low temperatures can be ascribed to crystallization through the formation of crystallites or clusters. Intensity light scattering measurements20 on aqueous solutions of β-CD have revealed that the size of the clusters rises with decreasing temperature and increasing concentration. This result corroborates well with the present findings. Temperature-induced crystallization transition of aqueous solutions of β-CD has been studied21 by differential scanning calorimetry, and the crystallization temperature was found to be slightly higher in D2O than in H2O. The temperature at which the first deviation of the scattered intensity from the baseline occurred was taken as the cloud point (CP) of the corresponding solution. The effect of β-CD concentration on CP is depicted in the inset plot of Figure 1. It is evident that the value of CP rises with increasing level of β-CD addition, and the value of CP may announce the formation of crystallites. It should be mentioned that the turbidity changes observed at low temperatures for the 2 and 4 mm samples could not be observed with the naked eye; thus, any crystallites, to the extent that they are formed, are very small. The turbidity curves in Figure 1 are the results from down-scans with a very slow cooling rate (0.05 °C/ min). However, if the sample after the down-scan is subsequently exposed to the same heating rate, a major hysteresis effect (not shown here), with much higher CP values than the corresponding ones during the down-scan, appears at high concentrations of β-CD. This signals that the gradual dissolution of the clusters in the subsequent heating cycle is a slow process. The turbidity features and kinetics have not been characterized here because it is beyond the scope of this work and some results in this direction have already been reported.20

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Figure 3. Comparison of the influence of temperature on the relative turbidity (divided by the value at 40 °C) for the alginate (2 wt %)/βCD/D2O and HM-alginate (2 wt %)/β-CD/D2O systems at different levels of β-CD addition. The size of the symbols indicates approximately the magnitude of the experimental error.

Figure 2. Effects of concentration of β-CD and temperature on the turbidity for 2 wt % solutions of alginate and HM-alginate dissolved in D2O. The turbidity was recorded at a wavelength of 500 nm.

The effects of β-CD addition on the turbidity of semidilute aqueous solutions of alginate and HM-alginate (2 wt %) on the turbidity at different temperatures are shown in Figure 2. The general trend is that the values of τ for low temperature and β-CD concentration higher than 4 mm are lower than the corresponding values in the absence of polymer. For the solutions of the unmodified alginate (Figure 2a), the turbidity rises only moderately with increasing β-CD concentration, and the turbidity change is most pronounced at the lowest temperature. Thus, it is clear that the dependencies of the turbidity features on β-CD concentration and temperature are significantly altered in the presence of alginate. This change in cloudiness of the solution is a sign of interaction between alginate and β-CD. In the presence of HM-alginate (Figure 2b), the changes in temperature and level of added β-CD have a more drastic influence on the turbidity. At the highest temperature, the value of τ decreases slightly as the amount of β-CD increases, whereas at the lowest temperature, the turbidity rises strongly with increasing β-CD concentration. The former finding suggests that, at elevated temperature, the hydrophobic moieties are progressively deactivated as the addition of β-CD increases, and this leads to weakening of the hydrophobic associations. The enhanced turbidity at lower temperatures with increasing β-CD concentration probably signals that only a small fraction of the hydrophobic sites of the polymer is encapsulated by β-CD and the excess of β-CD in the bulk forms crystallites. To facilitate a direct comparison between the temperatureinduced turbidity changes at various levels of β-CD addition for alginate and HM-alginate, the relative turbidity τ/τ40 (where τ40 is the turbidity at 40 °C) is plotted as a function of temperature in Figure 3. At β-CD concentrations up to 8 mm, the difference in temperature dependence of the relative turbidity for alginate and HM-alginate is small. However,

as the amount of added β-CD increases, a gradually stronger deviation between the turbidity curves appears at low temperatures. This seems to indicate that the interaction between alginate and β-CD prevents the growth of β-CD clusters in the bulk, whereas for HM-alginate only a few hydrophobic groups are decoupled, and because of steric hindrance, crystallites of β-CD are formed in the bulk at low temperatures instead of forming inclusion complexes with the polymer. The hypothesis is that steric hindrance from the hydrophobic groups attached on the polymer chains makes it difficult for the β-CDs to interact with the alginate backbone, leading to the formation of β-CD crystallites in the bulk at low temperature. In this context, we should notice (se the discussion below) that the capacity of β-CD to encapsulate hydrophobic groups is strongly reduced at low temperatures for the present system. Another possible scenario is that the hydrophobic moieties act as nucleation centers and thereby provide growth surfaces for further crystallization. Rheological Features. To minimize the influence of trivial changes of the solvent viscosity with temperature, the shear viscosity results are presented in terms of the relative viscosity ηrel (ηrel ≡ η/ηsolvent, where ηsolvent is the viscosity of heavy water). The effects of β-CD addition and temperature on the relative shear viscosity for the alginate/β-CD and HM-alginate/β-CD systems are depicted in Figure 4. A prominent feature for the alginate/β-CD system is the gradually more pronounced upturn of the relative viscosity at low shear rates as the temperature is lowered and the β-CD concentration is increased. The large viscosity enhancement seen in Figure 4 is a sign of that, at low temperature in the presence of a sufficient amount of β-CD, this cosolute forms clusters or crystallites that act as cross-linker of the alginate chains. These junction zones, formed through the interaction with β-CD clusters, are disrupted at high shear rates. Since no enhancement of the relative viscosity is visible at 40 °C, it is reasonable to assume that the β-CD clusters must be of a certain size to function as a cross-linker for the network. This conjecture is supported by the results in Figure 2a,

Effects of β-CD on an Associative Alginate

Figure 4. Effects of β-CD concentration and temperature on the shear rate dependence of the relative viscosity for 2 wt % solutions of alginate and HM-alginate dissolved in D2O. Every fourth point is shown to make the illustrations more transparent. The size of the symbols indicates approximately the magnitude of the experimental errors.

where the turbidity rises with decreasing temperature and increasing β-CD concentration. This observation suggests the formation of large aggregates. Furthermore, a dynamic light scattering study20 on aqueous solutions β-CD reveals that the clusters grow with decreasing temperature and increasing level of β-CD addition. This finding substantiates the idea that low temperatures and high β-CD concentrations promote the growth of large clusters. The cross-linkers are physical, and probably clusters are stabilized through hydrogen bonds, and the temperature-induced changes are reversible. The structure of the cross-linker zones and the mechanism of the cross-linking process are not known now. A different picture emerges for the HM-alginate/β-CD system, where a small effect of β-CD concentration is observed at 5 °C and a drastic drop of ηrel with increasing levels of β-CD addition is evident at 40 °C at low shear rates. In Figure 5a, the temperature and β-CD concentration dependencies of the relative zero-shear viscosity ηrel0 for the alginate/β-CD system are illustrated. It should be noted that the viscosity of β-CD solutions without polymer is close to that of D2O (ca. 10-3 Pas) at all conditions of temperature and β-CD concentration. At high levels of β-CD addition, a lowering of the temperature leads to the formation of large aggregates that sediment. In the considered temperature interval, the results at low temperatures reveal virtually no cross-linker effect at concentrations of β-CD below 8 mm. The enhancement of η0rel is shifted toward higher temperatures as the β-CD concentration increases and the effect become more accentuated. This novel finding announces that the interaction between β-CD and a polymer may not only lead to weakening of associations, but a strong network can be built. At the highest level of β-CD addition, the relative

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Figure 5. Effects of β-CD concentration and temperature on the relative zero shear viscosity for 2 wt % solutions of alginate and HMalginate dissolved in D2O.

zero-shear viscosity increases by almost three decades as the temperature decreases to 5 °C. Before we discuss the effects of β-CD concentration and temperature on the relative zero-shear viscosity for mixtures of HM-alginate and β-CD, it may be instructive to give some aspects on the influence of β-CD concentration and temperature on the deactivation of hydrophobic sites. For this purpose, we resort to a model developed by Karlson et al.22 in their rheological investigation of the interaction between cyclodextrins and hydrophobically modified polymers. In this simple approach, based on the Langmuir adsorption model, the β-CD molecules are regarded to bind to the hydrophobic tails of the polymer chains with a complex formation constant K. The hydrophobic attraction between the hydrophobic cavities (contain C-H groups) of β-CD and hydrophobic tails of the polymer drives a complex formation. In this model, the viscosity enhancement is considered to originate from associations via the polymer hydrophobic moieties, and the effect of entanglements is neglected. From this analysis, the following expression was obtained:22 η - η∞ )1-Θ)1η0 - η∞

[

12 B + cβ-CD + B + cβ-CD + 1/K K - Bcβ-CD 2 4 B

(

)

]

1/2

(1)

where η0 and η∞ are the zero-shear viscosity without β-CD and in excess of β-CD, respectively, Θ is the fraction of occupied binding sites in the Langmuir model, B is the concentration of polymer hydrophobic tails, and cβ-CD is the total concentration of β-CD. At each β-CD concentration, a value of Θ was determined from eq 1 with K and B as fitting parameters. The results from this fitting procedure are depicted in Figure 6, where the solid curves only connect

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Figure 6. Plot of Θ (fraction of occupied binding sites in the Langmuir model) as a function of the β-CD concentration for 2 wt % solutions of HM-alginate at different temperatures. At each β-CD concentration, a value of Θ was determined from eq 1 with K and B as fitting parameters. The curves only connect the points calculated from the fitting procedure. The fitting parameters B and K are displayed in the inset.

the data points to each other. The value of Θ rises with increasing β-CD concentration and a temperature raise yields higher values of Θ. This suggests that high levels of β-CD addition and elevated temperature promote the decoupling of hydrophobic interactions. The temperature dependencies of the fitting parameters are displayed in the inset plot. The value of B is practically independent of temperature, and this is expected because the number of polymer hydrophobic moieties should not be affected by temperature. The value of K increases with increasing temperature and this portends a more efficient complex formation between the hydrophobic tails and β-CD. Figure 5b shows the temperature dependence of the relative zero-shear viscosity for the HM-alginate/β-CD system at different levels of β-CD addition. A different behavior appears in this case compared to the alginate/βCD system. At high concentrations of β-CD (12 or 16 mm), η0rel falls off strongly with increasing temperature. This demonstrates clearly that high concentrations of β-CD and elevated temperature favor the decoupling of the polymer hydrophobic groups. In this process, the cavities of the β-CD structures make it possible for the hydrophobic tails of the polymer to reside inside them and form inclusion complexes with the β-CD molecules. A comparison of the β-CD concentration dependence of the zero-shear viscosity for 2 wt % solutions of alginate and HM-alginate at different temperatures is depicted in Figure 7. At the lower two temperatures (5 and 15 °C), no or only a slight decrease of the zero-shear viscosity can be traced for the HM-alginate sample as the level of added β-CD increases, suggesting that the decoupling of the hydrophobic sites is virtually absent. This indicates that the host-guest affinity decreases as the temperature is lowered. Since the turbidity of the system at low temperatures exhibits a drastic rise at higher levels of β-CD addition (cf. Figure 2), it is likely that low temperatures favor the growth of large β-CD clusters in the bulk and only a small fraction of β-CD molecules participates in the formation of complexes with

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Figure 7. Comparison of the β-CD concentration dependence of the zero-shear viscosity for the alginate (2 wt %)/β-CD/D2O and HMalginate (2 wt %)/β-CD/D2O systems at the temperatures indicated.

the polymer hydrophobic moieties. The reason is probably that at low temperatures, CDs are arranged in a “herringbone” pattern where the cavity of one molecule is blocked on both sides by adjacent, symmetry-related CD molecules, and this structure is not capable of encapsulating the hydrophobic moieties. On the other hand, we note that, at the same low temperatures (5 and 15 °C), the zero-shear viscosity for the solutions containing alginate shows a marked rise as the β-CD concentration increases. This effect may be ascribed to a situation where polymer chains are cross-linked via the assembling of β-CD molecules into complexes at junction zones. We note that the viscosity enhancement occurs at a higher level of β-CD addition at 15 °C than at 5 °C, and this strengthens the conjecture that the β-CD clusters must be sufficiently large to be efficient in the cross-linking of polymer chains. At higher temperatures (30 and 40 °C), the crystallization process is arrested and no β-CD concentrationinduced viscosification effect can be traced for the alginate solutions. This implies that the strength of the transient network formed in the semidilute alginate solution is not affected by β-CD addition, and this may be a sign of that the interaction between β-CD and alginate is suppressed at higher temperatures. For the solutions of HM-alginate, the zero-shear viscosity drops strongly with an increasing amount of β-CD at elevated temperatures. This signals weaker network structures due to the reduction of the number of active hydrophobes. As demonstrated above, the ability of β-CDs to form inclusion complexes with the attached hydrophobic groups of the polymer increases with increasing temperature, and this is reflected by the almost 30 unit dropz of the viscosity at 40 °C. At the concentration of 16 mm β-CD, it is interesting to note that at 30 °C only a fraction of the hydrophobes has been decoupled, whereas at 40 °C, almost all of the active hydrophobic tails are deactivated. The temperature effect can be rationalized in the following scenario. At elevated temperatures and in the absence of β-CD, the enhanced mobility of the polymer chains favors the growth of intermolecular hydrophobic interactions, as suggested by the increase of the relative viscosity with increasing temperature (cf. Figure 5b). During this process, the hydrophobic moieties

Effects of β-CD on an Associative Alginate

Figure 8. Effects of temperature and addition of β-CD on the dynamic moduli (ω ) 1.0 rad/s) for the alginate (2 wt %)/β-CD/D2O and HMalginate (2 wt %)/β-CD/D2O systems.

of the polymer become more accessible to β-CD encapsulation, and thereby the intensity of the hydrophobic association is reduced as the β-CD concentration increases. On the other hand, at lower temperatures some hydrophobic tails may be inaccessible to the β-CD molecules due to steric hindrance. Effects of temperature and β-CD concentration on the storage modulus (G′) and the loss modulus (G′′) for 2 wt % solutions of alginate and HM-alginate are shown in Figure 8. This illustration is constructed to elucidate the interplay between elastic and viscous responses in these systems at various conditions. At low additions of β-CD (0-4 mm), it is observed that G′ < G′′ for solutions containing alginate, whereas G′ > G′′ for mixtures with HM-alginate over the considered temperature range. These results demonstrate that the alginate/β-CD system exhibits a typical viscous behavior, whereas for the HM-alginate/β-CD system the elastic response dominates because of enhanced hydrophobic associations. Furthermore, at these low amounts of β-CD, the dynamic moduli fall off with increasing temperature for the alginate/β-CD systems, whereas G′ and G′′ both increase slightly as the temperature increases for the HM-alginate/ β-CD mixtures. Again these findings at low levels of β-CD (0-4 mm) addition support the conjecture that the higher chain mobility at elevated temperatures favors a weakening of the alginate network and the enhanced exposure and collisions of the hydrophobic groups lead to strengthening of the HM-alginate network. At higher levels of β-CD addition (>4 mm), a significantly different picture emerges for the HM-alginate systems, with a gradually stronger drop of the dynamic moduli with temperature as the β-CD concentration increases. The transient network theory23 predicts that the elastic modulus is directly proportional to the number of active junctions, and in light of this, the elastic response is expected to decrease as an increasing number of active hydrophobes is deactivated. The most prominent feature for the alginate/β-CD system at these conditions is

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Figure 9. SANS intensity (every third point is shown) plotted versus the scattering vector q at various β-CD concentrations and at different temperatures for the HM-alginate (2 wt %)/β-CD/D2O system. The size of the symbols indicates approximately the magnitude of the experimental error.

the strong upturn of G′ at low temperatures, reflecting the evolution of stronger network structures as a result of the cross-linking process. SANS Results. In the SANS experiments reported in this study, structures on a mesoscopic length scale are probed, whereas the turbidity (discussed above) is associated with a macroscopic length scale. Figure 9 shows the scattering spectra for 2 wt % HM-alginate solutions with different levels of β-CD addition at various temperatures. It should be noted that with this SANS spectrometer a restricted part of the low q region is probed. Therefore, detailed information in the Guinier regime is not obtained for this system. The intensity increase at low q depicts the scattering from some large structural inhomogeneities. We believe these to result either from molecular associations or from remaining hydrophobic interactions. It is apparent that in the intermediate q-range the curves at all conditions of temperature and β-CD concentration display an intensity that varies approximately as q-1. The q dependence of the scattered intensity at intermediate q values for this system at 25 °C has been reported previously.13 This behavior is characteristic of linear scattering arrays, as, for instance, may be expected from polymers that are locally stretched.24 The SANS spectra exhibit a similar profile at all conditions, and no conspicuous divergence in behavior, as in the case of the turbidity results (cf. Figure 2), can be discerned. Thus, the major macroscopic changes observed in the turbidity measurements are not reflected on the mesoscopic scale monitored in the SANS experiments. However, a close inspection of the scattering curves in the intermediate q region reveals that the effect of β-CD addition is small at 5 °C, whereas at higher temperatures, the values of the scattered intensity decrease somewhat as the β-CD concentration increases. The conjecture is that the difference in scattering behavior with temperature

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can partially be related to the intensity of the hydrophobic interactions. At 5 °C, virtually no decoupling of the hydrophobic sites occurs with increasing β-CD addition, whereas at higher temperatures, the progressive deactivation of the hydrophobic moieties as the percentage of β-CD rises leads to less polymer association complexes in the solution. Conclusions In this study, we have examined the effects of β-CD addition and temperature on the thermodynamic, structural, and rheological properties of semidilute solutions of alginate and a hydrophobically modified analogue. It is found that the cloud point of the β-CD solutions in the absence of polymer increases with the β-CD concentration. The effect of increasing β-CD addition and decreasing temperature on the turbidity enhancement of the alginate/β-CD/D2O system is moderate, whereas a much stronger response is observed for the HM-alginate/β-CD/D2O system. This shows that a high level of β-CD addition and a low temperature promote the formation of large-scale aggregates or crystallites in the presence of HM-alginate. The SANS results in the intermediate q range revealed that the HM-alginate chains are locally stretched (q-1 behavior). High levels of β-CD addition and reduced temperatures favor the growth of the relative zero-shear viscosity in alginate solutions, which is ascribed to the formation of a cross-linked network. In HM-alginate solutions with low concentration of β-CD, a temperature rise leads to higher values of the relative zero-shear viscosity, whereas at high levels of β-CD addition a temperature increase results in lower values of the relative viscosity. The augmented chain mobility at elevated temperatures facilitates the availability of hydrophobic sites for further intermolecular associations at low β-CD addition, hence the viscosity enhancement, whereas at high β-CD concentrations the encapsulation of hydrophobic moieties gives rise to a reduction of the viscosity. The viscosity data could be rationalized in the framework of a simple approach, based on the Langmuir adsorption model with the assumption that each polymer hydrophobic site/β-CD-complex decouples one rheologically active link in the polymer network.

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Acknowledgment. B.N., G.T.M.N., and K.D.K. gratefully acknowledge support from the Norwegian Research Council through a NANOMAT Project (158550/431). K.D.K. and C.G. thank the Marie Curie Industry Host Project (Contract No. G5TR-CT-2002-00089) for support. References and Notes (1) Goddard, E. D. In Interactions of Surfactants with Polymers and Proteins; Goddard, E. D., Ananthapadmananabhan, K. P., Eds.; CRC Press: Boca Raton, FL, 1993; p 123. (2) Brackman, J. C.; Engberts, J. B. F. N. Chem. Soc. ReV. 1993, 85, 1. (3) Lindman, B.; Carlsson, A.; Gerdes, S.; Karlstro¨m, G.; Piculell, L.; Thalberg, K.; Zhang, K. In Food Colloids and Polymers: Stability and Mechanical Properties; Walstra, P., Dickinson, E., Eds.; The Royal Society of Chemistry: London, 1993; pp 113-125. (4) Macromolecular Complexes in Chemistry and Biology; Dubin, P., Bock, J., Davies, R. M., Schulz, D. N., Thies, C., Eds.; SpringlerVerlag: Berlin, 1994. (5) Polymer-Surfactant Systems; Kwack, J. C. T., Ed.; Marcel Dekker: New York, 1998; Vol. 77. (6) Malmsten M. Surfactants and Polymers in Drug DeliVery, Drugs and the Pharmaceutical Sciences; Marcel Dekker: New York, 2002; Vol. 122. (7) Harada, A. Coord. Chem. ReV. 1996, 148, 115. (8) Harada, A. AdV. Polym. Sci. 1997, 133, 140. (9) Harada, A. Carbohydr. Polym. 1997, 34, 183. (10) Zhang, H.; Hogen-Esch, T. E.; Boschet, F.; Margaillan, A. Langmuir 1998, 14, 4972. (11) Sandier, A.; Brown, W.; Mays, H.; Amiel, C. Langmuir 2000, 16, 1634. (12) Saenger, W. Jerusalem Symp. Quantum Chem. Biochem; Pullman, E. B., Ed.; Riedel, Co.: Dordrecht, The Netherlands, 1976. (13) Galant, C.; Kjøniksen, A.-L.; Nguyen, G. T. M.; Knudsen, K. D.; Nystro¨m, B. J. Phys. Chem. B, submitted for publication. (14) Nakajima, N.; Ikada, Y. Bioconjugate Chem. 1995, 6, 123. (15) Hermanson, G. T. In Bioconjugate Chemistry; Academic Press: San Diego, CA, 1996; p 169. (16) Park, S. N.; Park, J.-C.; Kim, H. O.; Song, M. J.; Suh, H. Biomaterials 2002, 23, 1205. (17) Kjøniksen, A.-L., Laukkanen, A.; Galant, C.; Knudsen, K. D.; Tenhu, H.; Nystro¨m, B. Macromolecules 2005, 38, 948. (18) Cotton, J.-P. J. Phys. IV France 1999, 9, 21. (19) Betzel, K.; Saenger, W.; Hingerty, B. E.; Brown, G. M. J. Am. Chem. Soc. 1984, 106, 7545. (20) Georgalis, Y.; Schu¨ler, J.; Umbach, P.; Saenger, W. J. Am. Chem. Soc. 1995, 117, 9314. (21) Frank, J.; Holzwarth, J. F.; Saenger, W. Langmuir 2002, 18, 5974. (22) Karlson, L.; Thuresson, K.; Lindman, B. Carbohydr. Polym. 2002, 50, 219. (23) Tanaka, F.; Edwards, S. F. Macromolecules 1992, 25, 1516. (24) Spiteri, M. N.; Boue, F.; Lapp, A.; Cotton, J. P. Phys. ReV. Lett. 1996, 77, 5218.

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