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Dextran Sulfate-Amphiphile Interaction; Effect of Polyelectrolyte Charge Density and Amphiphile Hydrophobicity B. Persson, A. Hugerth, N. Caram-Lelham,* and L.-O. Sundelo¨f Physical Pharmaceutical Chemistry, Uppsala University, Uppsala Biomedical Center, Box 574, S-751 23, Uppsala, Sweden Received June 4, 1999. In Final Form: August 16, 1999 The interaction between dextran sulfate (DxS), of different charge density, and three cationic amphiphiles varying with respect to hydrophobicity, was investigated by means of equilibrium dialysis and surface tension. The hydrophobicity of the amphiphilies studied increases in the order doxepin-HCl < amitriptylineHCl < clomipramine-HCl. Dextran sulfate samples with five different charge densities, 0.07, 0.43, 0.7, 1.3, and 1.6 charges per monosachride, were used. The data from both methods clearly indicate that the polyelectrolyte-amphiphile interaction shows a cooperativity that starts at a certain amphiphile concentration and which depends on both the hydrophobicity of the amphiphile and the charge density of the dextran sulfate. A change in the charge density of dextran sulfate and/or amphiphile hydrophobicity affects both the onset of the cooperativity of the interaction and the degree of the cooperativity. The results have also been compared with the data published on related systems.
Introduction The strength and the nature of the interaction between water soluble polyelectrolytes and oppositely charged amphiphiles depends on the characteristics of both the polyelectrolyte and the amphiphile. With respect to the polyelectrolyte, factors such as charge density,1-3 flexibility,4 conformation,5 type of charges,6 degree of polymerization (Mw),7 and the presence and distribution of hydrophobic and hydrophilic segments8-11 are decisive. Studies of the interaction of charged polysaccharide/ amphiphilic molecules1-5,12-14 have clearly demonstrated the importance of the charge density parameter. However, the effect of charge density on the polyelectrolyteoppositely charged amphiphile interaction is often difficult to isolate from other factors, especially the hydrophobic interaction between the amphiphile and the polyelectrolyte.15,16 In the present work a series of sodium dextran * To whom correspondence should be addressed. Tel.: +46-184714368. E-mail:
[email protected]. (1) Caram-Lelham, N.; Sundelo¨f, L.-O. Int. J. Pharm. 1995, 115, 103111. (2) Hansson, P.; Almgren, M. J. Phys. Chem. 1996, 100, 9038-9046. (3) Malovikova, A.; Hayakawa, K.; Kwak, J. C. ACS Symp. Series 1984, 253, 225-239. (4) Caram-Lelham, N.; Hed, F.; Sundelo¨f, L.-O. Biopolymers 1997, 41, 765-772. (5) Caram-Lelham, N.; Sundelo¨f, L.-O. Biopolymers 1996, 39, 387393. (6) Hansson, P.; Almgren, M. J. Phys. Chem. 1995, 99, 16694-16703. (7) Liu, J.; Shirahama, K.; Miyajima, T.; Kwak, J. C. T. Colloid Polym. Sci. 1998, 276, 40-45. (8) Anthony, O.; Raoul, Z. Langmuir 1996, 12, 1967-1975. (9) Anthony, O.; Raoul, Z. Langmuir 1996, 12, 3590-3597. (10) Hansson, P.; Almgren, M. Langmuir 1994, 10, 2115-2124. (11) Schimizu, T.; Kwak, J. C. T. Colloids and Surf. A: Physicochemical and Engineering Aspects 1994, 82, 163-171. (12) Caram-Lelham, N.; Sundelo¨f, L.-O. Pharm. Res. 1996, 13, 920925. (13) Persson, B.; Caram-Lelham, N.; Sundelo¨f, L.-O. Int. J. Biol. Macromol. 1996, 19, 263-269. (14) Singh, S. K.; Caram-Lelham, N. J. Colloid Interface Sci. 1998, 203, 430-446. (15) Hayakawa, K.; Kwak, J. C. T. J. Phys. Chem. 1982, 86, 38663870. (16) Kiefer, J. J.; Somasundaran, P.; Ananthapadmanabhan, K. P. Langmuir 1993, 9, 1187-1192.
sulfate (DxS) samples which differ only with respect to charge density, was employed. The set of DxS samples used are well suited for a comparative discussion concerning the influence of the charge density on the amphiphile adsorption since they are flexible, hydrophilic, and synthesized from dextran of the same MW, 70 000 (except for the DxS with the charge density of 0.43 which has a molecular weight of 150 000). Some of the amphiphile characteristics affecting the interaction are: hydrophobicity,17,18 rigidity and bulkiness of the apolar part and the position and type of charge of the polar part. In the present study the amphiphiles, clomipramine-HCl (3-chloro-10,11-dihydro-N,N-dimethyl5H-dibenz[b,f]azepine-5-propanamine), amitriptyline-HCl (3-(10,11-dihydro-5H-dibenz[a,d]-cyclohepten-5-ylidene)N,N-dimethyl-1-propanamine) and doxepin-HCl (3-dibenz[b,e]oxepin-11(6H)-ylidene-N,N-dimethyl-1-propanamine), constitute a group of amphiphilic drug molecules, (Figure 1), which are used mainly for treatment of the symptoms of depression. The hydrophilic part of the amphiphiles are identical but they differ with respect to the hydrophobic part, a three cyclic ring structure. These amphiphiles have rather bulky and rigid apolar parts and do not, in contrast to surfactants such as SDS and C12TAB, form traditional spherical micelles but rather nonspherical aggregates with low aggregation numbers.19,20 Thus, the amphiphiles used in this study not only facilitate an evaluation of the influence of the amphiphile hydrophobicity on the polyelectrolyte-amphiphile interaction but also illustrate how amphiphilic substances, with similar structure to those employed, interact with polyelectrolytes. Investigations of polyelectrolyte-amphiphile interaction have utilized a wide variety of techniques such as: equilibrium dialysis,1,4,5,12 surfactant ion-selective electrodes,15 surface tension measurements (pendant drop),13 (17) Malovikova, A.; Hayakawa, K.; Kwak, J. C. T. J. Phys. Chem. 1984, 88, 1930-1933. (18) Okuzaki, H.; Osada, Y. Macromolecules 1994, 27, 502-506. (19) Attwood, D.; Gibson, J. J. Pharm. Pharmacol. 1978, 30, 176180. (20) Moro, M. E.; Rodriguez, L. J. Langmuir 1991, 7, 2017-2020.
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Figure 1. Primary structure formulas of amphiphiles.
microcalorimetry,14,21 dynamic light scattering,22 and fluorescence spectroscopy (steady state and time-resolved),23 to mention a few. Equilibrium dialysis and surface tension measurements were chosen for this study since they offer a straightforward and rapid means of obtaining fundamental data describing the system at hand. The results will be discussed according to two models commonly used to analyze the polyelectrolyte-amphiphile interaction. The first part of the discussion analyzes the polyelectrolyte-amphiphile interaction qualitatively by the nearest-neighbor interaction model.24 This model considers mainly the hydrophobic interaction between amphiphiles adsorbed to a linear array of charged binding sites provided by the polyelectrolyte. In the second part, the results are examined according to the polyelectrolyteinduced micelle formation model,21,25 which focuses on the effect of the polyelectrolyte charge density. Experimental Section Materials. Dextran sulfate samples were kindly provided by Pharmacia & Upjohn Inc. (Uppsala, Sweden), except for the sample with a charge density of 0.43 (number of charges per saccharide unit) which was a gift from TdB Consultancy AB (Uppsala, Sweden). The molecular weight of the dextran used for the synthesis of dextran sulfates, received from Pharmacia & Upjohn Inc. and from TdB Consultancy AB were approximately 70 000 and 150 000, respectively. Clomipramine-HCl, amitriptyline-HCl, and doxepin-HCl were purchased from Sigma (St. Louis, MO). The polymer and amphiphile samples were used as received. The dextran sulfate used in this study had the following charge densities, defined as the average number of charged groups per monosaccharide unit: 0.07, 0.43, 0.7, 1.3, and 1.6. Since the charge group on the dextran is sulfate and the pKa of amphiphiles is much higher than the pH of the solutions, which is close to neutrality, both the polyion and the amphiphile are fully charged during our experiments. All other chemicals were commercially available products of analytical grade. Dialysis Equilibrium. The degree of binding of the amphiphile to the polymer was determined by a previously described dialysis equilibrium method utilizing specially designed Lucite dialysis cells.1 All measurements where carried out at ambient temperature and lasted some 24 h, which was shown to be (21) Skerjanc, J.; Kogej, K.; Vesnaver, G. J. Phys. Chem. 1988, 92, 6382-6385. (22) McQuigg, D. W.; Kaplan, J. I.; Dubin, P. L. J. Phys. Chem. 1992, 96, 1973-1978. (23) Hansson, P.; Almgren, M. J. Phys. Chem. 1995, 99, 1668416693. (24) Sateke, I.; Yang, J. T. Y. Biopolymers 1976, 15, 2263-2275. (25) Abuin, E. B.; Scaiano, J. C. J. Am. Chem. Soc. 1984, 106, 62746283.
Figure 2. The effect of charge density 0.43 (9), 0.7 (B), 1.3 (4), 1.6 (2) on binding isotherms for (a) clomipramine, (b) amitriptyline, and (c) doxepin, in 0.5 mg/g DxS and 30 mM NaCl. sufficient to attain dialysis equilibrium. The dialysis membrane was of a regenerated cellulose, purchased from Spectrum (CA), with a molecular cutoff 12 000-14 000. In a previous study, it was shown that the adsorption of drug to these membranes was negligible.1 The concentration of amphiphile was determined spectrophotometrically. DxS did not affect the spectral properties of the drug molecules at the measuring wavelengths. Surface Tension Measurements. The samples for the surface tension measurements where prepared in carefully cleaned glassware and the solutions where protected from exposure to light. The surface tension measurements were performed by a du Nou¨y tensiometer (A. Kru¨ss OptischMechanische Werksta¨tten, Hamburg, Germany) against air at 25 °C. All measurements where carried out not earlier than 10 min after formation of a new surface.13,26 In separate experiments, it was shown that the period of 10 min was sufficient to ensure that an apparent steady-state surface tension, γ*, was reached.
Results and Discussion I. Adsorption Isotherms and Surface Tension Curves. Binding isotherms, given in Figures 2 and 4a, show the degree of binding, β, versus free amphiphile (26) Schwuger, M. J. J. Colloid Interface Sci. 1973, 43, 491-498.
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Figure 3. Surface tension versus concentration of doxepin ([D+]tot) for DxS (0.1 mg/g) of linear charge density 0.07 (n), 0.43 (9), 0.7 (B), 1.3 (4), and 1.6 (2) in 6 mM NaCl.
Figure 4. Effect of hydrophobicity of the amphiphile doxepin (B), amitriptyline (4), and clomipramine (2), respectively, on (a) the binding isotherms and (b) surface tension curves for DxS with the charge density 0.43.
concentration [D+]I. β designates the number of moles of amphiphile adsorbed per mole polymer charge and is defined by the relation
β ) ([D+]II tot - [D+]I)/Cp*
(1)
[D+]II tot is the total concentration of amphiphile in the compartment containing polymer and [D+]I is the free amphiphile concentration in the compartment without polymer. Cp* is the number of moles of polymer charges as calculated from the polymer mass concentration, the Mw of the monosaccharide unit, and the charge density. The polymer and salt concentrations in all sample solutions were 0.5 mg/g and 30 mM (NaCl), respectively. The Donnan effect was considered to be almost negligible because of the very low polymer concentration compared to the salt concentration present. The apparent steadystate surface tension, γ*, versus the total amphiphile
concentration, at a polymer concentration of 0.1 mg/g and a NaCl concentration of 6 mM, is presented in Figures 3 and 4b. Typical surface tension data as a function of charge density are represented by Doxepin in Figure 3. All surface tension experiments were performed at a lower polymer concentration than the equilibrium dialysis experiments to diminish the risk of precipitation of the polymer/ amphiphile complex. To keep the concentration ratio between the polymer and salt constant, salt concentration was also decreased. The adsorption isotherms and surface tension measurements were performed up to the amphiphile concentration where precipitation of the complex begins. The features of the curves in Figures 2 and 3 are similar; at low amphiphile concentration the change in surface tension and the degree of binding is very low. At a certain amphiphile concentration the slopes of the curves change drastically. It is obvious that there is a relationship between the degree of adsorption and the decrease in surface tension. In the present case the adsorption of charged amphiphiles to oppositely charged polymers at low amphiphile concentration is due to charge-charge interaction. However, at higher amphiphile concentration this is not the only mechanism. When the concentration of amphiphile reaches a particular value, defined as the critical aggregation or cooperativity concentration, cac, a strong increase in adsorption and a clear decrease in surface tension occur. This cooperativity has been shown to be due to the hydrophobic interaction between the apolar parts of the already adsorbed amphiphiles.17 It is important to mention that the concentrations of the amphiphiles in the solutions studied here are far below the cmc values. Therefore the decrease in the surface tension in the amphiphile/polyion system can only be explained by the formation of surface active complexes. Goddard27 has also shown that any synergistic lowering of surface tension in solutions containing polymer and amphiphile indicates an interaction between these molecules. In the composition range of our experiments it is very unlikely that free micelles exist. However, the bound amphiphile molecules may form some type of aggregates along the polymer chain. The degree of cooperativity has, in the literature,15,24 been estimated from the slopes of the binding isotherms at the half bound point. Due to precipitation of the polyionamphiphile complex at low β-values, an adequate quantitative estimation of the cooperativity cannot be performed appropriately.24 However, the data provide a means for qualitative discussion of the cooperativity. Figures 2 and 3 show that an increase in charge density over 0.43 drastically decreases the cac and increases the slopes of the curves. At the highest charge densities, 1.3 and 1.6, the slopes are almost vertical indicating a very high cooperativity. The high cooperativity is mainly due both to a short charge-charge distance along the polymer and a high flexibility of the polymer. A decrease of the distance between the charges on the polymer leads to an increased interaction between the hydrophobic moieties of the adsorbed neighboring amphiphile molecules. The data reveal that already at a charge density of 0.7 the charge-charge distance is short enough to allow a strong hydrophobic interaction between adjacent amphiphiles to occur. A higher charge density (1.3 and 1.6) further increases the cooperativity. The adsorption isotherms for charge density 0.07 are not shown due to the low degree of binding in this case. (27) Goddard, E. D. Interactions of Surfactants with Polymers and Proteins; Goddard, E. D., Ed.; CRC Press: Boca Raton, FL, 1993; Chapter 4, pp 171-201.
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The amphiphiles chosen for this study (Figure 1) have the same hydrophilic part which is a tertiary amine. This similarity is reflected in the very small difference in their pKa values, 9.0-9.5.28 The structural differences between these amphiphiles reside in the apolar part, which thus determines the degree of hydrophobicity and is reflected in the critical micelle concentration (cmc). The cmc values of doxepin-HCl, amitriptyline-HCl, and clomipramine-HCl are 60, 28, and 14 mM, respectively, as determined by the pendant drop method in 6 mM NaCl.13 Thus, the hydrophobicity increases in the order doxepin-HCl < amitriptyline-HCl < clomipramine-HCl. As mentioned above, the amphiphiles are fully charged in the solution used in our experiments due to their high pKa values. Figures 4a and 4b show the adsorption isotherms and γ* versus amphiphile concentration for three different amphiphiles in DxS with charge density 0.43. The data presented in these figures clearly indicate that the differences in hydrophobicity of the apolar parts of the amphiphiles affect the degree of binding. From the adsorption isotherms it is obvious that an increase in hydrophobicity gives a decrease in cac as well as an increase of the slope of the isotherms, thereby indicating a higher degree of cooperativity. The γ* curves show almost the same general pattern where the breakpoint concentration decreases with increasing hydrophobicity. The high degree of flexibility of DxS facilitates the hydrophobic interaction between neighboring amphiphiles on the polymer. This was confirmed in an earlier study, where dextran sulfate was compared with κ-carrageenan and hyaluronan of the same charge density. DxS showed a much higher cooperativity compared to the other two polymers which are known to be less flexible.4 The isotherms shown in Figures 2 and 4a indicate that precipitation occurs long before the degree of binding, β, reaches unity. Adsorption of amphiphile to the polyelectrolyte not only partially neutralizes the charges of the polymer but also results in the formation of hydrophobic complexes, which render the polyelectrolyte insoluble. The DxS-amphiphile complexes formed phase separate at lower β-values than those normally observed with traditional surfactants15 emphasizing the dependence of the polyelectrolyte-amphiphile interaction on the surfactant characteristics. Compared to other charged polysaccharides such as κ-carrageenan, alginate, and hyaluronan,4 higher β-values are reached in the DxS case. This can be explained by the hydrophilicity of DxS which is not only due to the charged groups on the polymer, but also because of the relatively hydrophilic backbone. II. Polyelectrolyte-Induced Amphiphile Aggregation Model. The discussion presented above was based on the nearest-neighbor interaction model24 but the interaction between amphiphile and polyion can also be modeled as a polyelectrolyte-induced amphiphile aggregation process.21,23,29 This approach is based on the well-known fact that an inorganic electrolyte is unevenly distributed in an aqueous polyelectrolyte solution.30-33 Adding an amphiphile to a solution of a polyelectrolyte of opposite charge will consequently result in an inhomogeneous distribution of the amphiphile as well. Thus, the concentrations of the inorganic electrolyte and amphiphile (28) Moffat, A. C.; Jackson, J. V.; Moss, M. S.; Widdop, B. Clarke’s Isolation and Identification of Drugs; Moffat, A. C., Jackson, J. V., Moss, M. S., Widdop, B., Eds.; The Pharmaceutical Press: London, 1986. (29) Lo¨froth, J.-E.; Johansson, L.; Norman, A.-C.; Wettsro¨m, K. Prog. Colloid Polym. Sci. 1991, 84, 78-82. (30) Beyer, P.; Nordmeier, E. Eur. Polym. J. 1995, 31, 1031-1036. (31) Bare, W.; Nordmeier, E. Polymer J. 1996, 28, 712-726. (32) Manning, G. S. J. Chem. Phys. 1969, 51, 924-933. (33) Manning, G. S. Q. Rev. Biophys. 1978, 11, 179-246.
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Figure 5. Effect of polyelectrolyte charge density on cac for clomipramine (9), amitriptyline (2), and doxepin (B), in 0.5 mg/g DxS and 30 mM NaCl. Corresponding values for κ-carrageenan (unfilled symbols) by Caram-Lelham et al.12 and the NaCMC/C12TAB system (×) by Hansson et al.2 are shown as a comparison.
are enhanced in the vicinity of the polyelectrolyte compared to the bulk phase.21,23,29 These factors combined will facilitate the formation of amphiphilic aggregates at concentrations (taken as an average of the total solution volume) significantly less than the cmc in a polyelectrolyte free solution. Hence the model briefly outlined above could be anticipated to depend critically on the effective charge density of the polyelectrolyte. Investigations of the effect of charge density of homologous series of a polyelectrolyte on the polyelectrolyte-amphiphile interaction are, to our knowledge, thus far scarce.2,3 A first approximation of the effective polyelectrolyte charge density is given by the reduced linear charge density parameter32 ξ, where ξ ) e2/(4π0rkTb) and e is the unit charge, 0r is the permittivity of the medium, k is Bolzmann’s constant, T is the absolute temperature, and b is the distance between charges. ξ has a critical value of 1.0, (ξcrit ) 1.0). If ξ of the polyelectrolyte exceeds ξcrit, counterions will condense on the polyelectrolyte to lower the reduced linear charge density to this value.32 Although DxS cannot be expected to behave strictly according to Manning’s limiting law at the conditions of the investigation,30,31 the reduced linear charge density parameter can be used as a relative measure within the homologous series with respect to charge density of the polyelectrolytes studied. The number of charges per monosaccharide (cd) of a polyelectrolyte is an easily obtainable quantity and provides a straightforward measure of ξ since it is directly proportional to ξ. Figure 5 shows the dependence of the onset of amphiphile aggregation, the critical aggregation concentration, cac (which is equivalent to ccc4 as well as T134), on the number of charges per monosaccharide of the polyelectrolyte. As the cd increases, the cac decreases sharply until the cd is approximately 0.75. The value 0.75 corresponds closely to ξcrit.30 An additional increase in cd affects the cac marginally. In addition, Figure 5 illustrates (as indicated above) that the cac decreases as the hydrophobicity of the apolar parts of the amphiphile increases. Data from an investigation of the interaction between κ-carrageenan and the same set of amphiphilic drug molecules supports the general trend indicated (Figure 5).12 A comparison of the dependence of cac on the charge density found in the DxS/amphiphilic drug molecule systems with the dependence found by Hansson et al. for the NaCMC/C12TAB system2 shows that both systems have common features with regard to ξcrit. This (34) Goddard, E. D. J. Am. Oil Chem. Soc. 1994, 71, 1-16.
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is noteworthy since the systems differ with respect to the type of charge on the polyelectrolyte and the physicochemical properties of the amphiphile. Furthermore, the dependence of cac on charge density found for the DxS/ amphiphile system agrees well with data presented for other polyelectrolyte-amphiphile systems although there is a wide discrepancy between the system characteristics.35 Concluding Remarks The adsorption isotherms and the surface tension curves show that the DxS-amphiphile interaction depends (35) Lindman, B.; Thalberg, K. Interactions of Surfactants with Polymers and Proteins; Goddard, E. D., Ed.; CRC Press: Boca Raton, FL, 1993; Chapter 5, pp 203-268.
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strongly on the linear charge density of the polyelectrolyte as well as of amphiphile hydrophobicity. An increase in charge density or/and amphiphile hydrophobicity results in a decrease of cac and an increase in the cooperativity of the adsorption process. The characteristics of the polyelectrolyte-amphiphile interaction and solubility of complexes formed is closely related to the structure of the amphiphiles. Acknowledgment. Financial support from the Swedish Natural Science Research Council and the Swedish Council for Engineering Sciences are gratefully acknowledged. LA990708V
