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Amphiphilic Derivatives of Alginate: Evidence for Intraand Intermolecular Hydrophobic Associations in Aqueous Solution A. Sinquin, P. Hubert,* and E. Dellacherie Laboratoire de Chimie-Physique Macromolbculaire, URA CNRS 494, ENSIC, B P 451, 54001 Nancy Cedex, France Received June 7, 1993. I n Final Form: September 13, 199P The covalent fixation of a long alkyl chain ( C ~ Zonto ) a partially esterified derivative of sodium alginate (PGA) affords an amphiphilic compound which exhibits in aqueous solution the typical properties of hydrophobically associatingwater-solublepolyelectrolytes. Rheological measurements carried out in the semidiluteregime evidence the occurrenceof intermolecular hydrophobic interactions,resulting in strongly enhanced viscosity. At high dilution, viscometric data suggest the formation of compact conformations, resultingfrom intramolecular hydrophobic associations. Fluorescenceexperiments conducted with pyrene showthat polymer concentrations as low as 0.05 5% are sufficientto create a hydrophobic microenvironment for this probe. The phenomenon is enhanced upon increasing the ionic strength.
Introduction Hydrophobicallyassociating water-soluble polymers are receiving increasing attention owing to their potential in various industrial or biotechnological applications. The physicochemical properties of these polymers in aqueous solution are directly related to the involvement of intra- or intermolecular associations between the hydrophobic moieties, according to the concentration range concerned. At high dilution, intramolecular attractions are favored and the polymer adopts a micelle-likemicrophase structure where the hydrophobic units form an interior hydrophobic domain that could be exploited, e,g. for the dissolution and transport of lipophilic drugs. In the semidilute regime, hydrophobic segments may undergo intermolecular association to form multimolecular clusters, resulting in strongly enhanced viscosity and peculiar rheological properties. Apart from a few articles dealing with the preparation of amphiphilic polyelectrolytes from polystyrene1 or maleic anhydride,24 most studies in this field have been carried out on a large variety of derivatives of acrylic acid. This monomer has been combined, under diverse hydrophilic versions (sodium acrylate, sodium (acry1amido)propanesulfonate, N-isopropylacrylamide, ...), with long-chain alkylamines,5p6surfactant macromonomers,' fluorocarbon derivatives,*or, for photophysical purposes, with various polyaromatic hydrocarbon~.~J~ To a lesser extent, polysaccharides have also been selected as hydrophilic backbones. Hydrodynamic properties of hydrophobically modified hydroxyethyl cellulose
* To whom correspondence should be addressed.
e Abstract published in Advance ACS Abstracts, November 15, 1993. (1) Itoh, Y.; Negishi, K.; Iizuka, E.; Abe, K.; Kaneko, M. Polymer 1992, 33, 3016.
(2) Mgtesh, C.; Xu,Q.; Somasundaran, P.; Benton, W. J.; Nguyen, H. Langmuir 1992,8, 1511. (3) Jye-Ling Hsu; Strauss, V. P. J. Phys. Chem. 1987, 91, 6238. (4) Strauss, V. P.; Vesnaver, G. J.Phys. Chem. 1975, 79, 1558. (5) Magny, B.; Lafuma, F.; Iliopoulos, I. Polymer 1992, 33, 3151. (6) Wang, K. T.; Iliopoulos,I.; Audebert, R. Polym.Bull. 1988,20,577. (7) Schulz, D. N.; Kaladas, J. J.; Maurer, J. J.; Bock, J.; Pace, S. J.; Schulz, W. W. Polymer 1987,28, 2110. (8) Seery, T. A. P.; Yassini, M.; Hogen-Esch, T. E.; Amis, E. J. Macromolecules 1992,25,4784. (9) Morishima, Y. Prog. Polym. Sci. 1990, 15, 949. (10) Ringsdorf, H.; Simon, J.; Winnik, F. M. Macromolecules 1992,25, 7306.
0743-7463/93/2409-3334$04.00/0
(HMHEC) have been described throughout the last decade, following the initial article by Landoll, in 1982.11 The spontaneous formation of nanoparticles from palmitoyl- or cholesterol-bearing pullulan and their ability to host various globular proteins may also be mentioned.I2 In the present article, we report our preliminary results on the introduction of hydrophobic aliphatic side chains in a derivative of sodium alginate. This study was effected with the ambition of going beyond the mere observation of uncommon physicochemical properties of the aqueous solutions. The alginate salts differ from most other polysaccharides in that they exhibit a sol-gel transition when simply submitted to modifications of their ionic environment, e.g. substitution of Na+ by divalent cations such as Ca2+. This property is, in ow opinion, an additional motivation for the preparation of amphiphilic macromolecules and their study, not only in solution but also, in the near future, in the gel state.
Experimental Section The starting derivative of alginate that we used was provided by Protan (Drammen, Norway). This compound, propylene glycol alginate (PGA), is produced on an industrial scale by treatment of alginicacid with propylene oxide. The esterification is not complete but, as claimed by the supplier, superior to 70% of the available carboxylic groups. The amphiphilic derivative (PGA-Cl2throughout the present article)was prepared after the procedure described by Yalpani and Hall.13 It consists of a nucleophilicdisplacement of the ester by dodecylamine. The reaction was carried out during 15 min in anhydrous dimethylformamide, at room temperature. The polymer was purified by precipitation in absolute ethanol, followed by extensive washings, successively with absolute ethanol, dioxane, and, finally, acetone, until a reliable constant nitrogen analysis is obtained. In the resulting PGA-CIZ, about 9% of the total saccharide units were substituted with dodecylamine, as determined by nitrogen analysis. Polymer solutionswere prepared from ultrapure water (MU-Q water purification system, Millipore) under vigorous stirring for 24 h. When necessary, solutions of salts at adequate concentrations were added to the previously prepared aqueouspolymer solutions. The resulting mixtures were stirred for a further 12 hand then allowed to stand for at least 24 h before measurements were performed. (11) Landoll, L. M. J. Polym. Sci. 1982, 20, 443. (12) Akiyoshi, K.; Nagai, K.; Nishikawa, T.; Sunamoto, J. Chem. Lett. 1992, 1727. (13) Yalpani, M.; Hall, L. D. Can. J. Chem. 1981,59, 3105.
0 1993 American Chemical Society
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Molecular weights, averages, and distributions, were determined by size exclusion chromatography (SEC) (TSK G 6000 column, Waters 590 HPLC pump) coupled to on-line dual refractometric (Waters 410) and low angle laser light scattering (LALLS) (KMXG, Milton-Roy, USA) detection. Viscometric measurements at high dilution were carried out with an Ostwald-type automatic capillary viscometer (0.36 mm diameter) (Viscologic TI.1, Sematech, France) thermostated at 25 f 0.05 O C . In the semidilute regime, viscosity measurements were per: formed with a ContraveeLS-30viscometer, at low shear rate (y = 0.06 d).The temperature (25 "C) was controlled to within 0.1 "C. Fluorescenceemission spectra of pyrene (1.1X 108 M) added to the considered polymer solutions were recorded in the 350500 nm range on a SPEX Fluorolog 2 spectrometer. The excitation wavelength was 335 nm.
Results and Discussion The immobilization of long aliphatic chains on sodium alginate can be envisaged either directly on hydroxyl or carboxyl groups of the uronic (guluronic and mannuronic) acids or after creation on the polysaccharidic backbone of new functionalities. The reactivity of hydroxyl groups is very weak and only the recently described acetylation proceeds successfully.14 Carboxylic acids can be condensed with desired amines after preliminary activation, e.g. with water-soluble carbodiimides such as l-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDCI).However, the activated intermediary 0-acylisourea may not only react as expected with the aliphatic amine to yield the desired amide but also isomerize to some extent into a stable N-acylurea15 and therefore lead to a derivative contaminated by indesirable pendant chains. Another alternative, consisting of the formation of dialdehydes by metaperiodate oxidation of vicinal diols, of the sugar units, is generally accompanied by an extensive depolymerization'B-l7which may result in a complete loss of the sol-gel transition properties. On the other hand, propylene glycol esters of alginate (PGA) can reportedly react mildly with a wide range of amines to form amides in high yields at ambient temperature.ls Nonesterified sodium carboxylates remain sufficient so that this derivative can retain not only the typical viscometric behavior exhibited by polyelectrolytes in dilute pure water solution but, above all, the ability of sol-gel transition upon treatment with Ca2+. This derivative thus appears as an adequate precursor for the synthesis of the desired gel-formingamphiphilic polymers. Hydrophobically associating water-soluble polyelectrolytes have been reported to behave quite differently according to the concentration range concerned. Above a certain critical concentration, the chains interact intermolecularly to form aggregates of large hydrodynamic volume and accordinglyvery high viscosity, whereas below this critical concentration, the chains interact intramolecularly, leading to shrunken conformations of the ~oi1.697911~19 Figure 1 shows the concentration dependence of the solution viscosity, for PGA and PGA-CIZ in pure water. (14) Skjak-Braek, G.;Paoletti,S.;Gianferrara,T. Carbohydr.Res.1989, 185, 119. (15)Papisov,M. I.;M&imenko, A.V.;Torchilin,V.P.EnzymeMicrob. Technol. 1985, 7,11. (16)Andresen, I. L.; Painter, T.; Smidsrod, 0. Carbohydr. Res. 1977, 59, 563. (17) Painter, T.; Larsen, B. Acta Chem. Scand. 1970,24, 813. (18) McDowell, R. H. J. SOC.C o m e t . Chem. 1970,21,441. (19) McCormick, L. C.; Nonaka, T.; Johnson, C. B. Polymer 1988,29, 731.
1
0.5
1.5
C (gW Figure 1. Viscosity of (0) PGA and ( 0 )PGA-CIZ, in pure water, vs polymer concentration (shear rate = 0.06 8-1).
+
,
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O
0
1
2
3
Figure 2. Viscosity of (0)PGA and ( 0 ) PGA-C12 vs NaCl concentration (C,) (polymer concentration c = 1.2 g/dL; shear rate i. = 0.06 s-l).
PGA exhibits a slow increase of its viscosity, in agreement with typical polyelectrolyte behavior when concentration is sufficiently high so that electrostatic repulsions are screened. In opposition, the viscosity of PGA-Cl2 sharply increases with the concentration, to reach values 2 orders of magnitude higher than those of the precursor. The hydrophobic nature of the phenomena observed is clearly evidenced by the effect of ionic strength on the respective viscosities of PGA and PGA-Clz. In the presence of salts (Figure 2) the viscosity of PGA-Cl2 is strongly enhanced, whereasthe unmodified polysaccharideis barely affected. These results can be interpreted in terms of intermolecular hydrophobic interactions between the dodecyl chains immobilized on PGA. As the polymer concentration increases, hydrophobic domain ordering becomes important and may ultimately result in a "pseudogel" structure, where associations are an intermediate between the transient polymeric entanglements found in semidilute solutions and permanent chemical cross-links in covalent gels or networks. When the polymer concentration is made sufficiently low, the chain may then be considered as isolated in the solution, free of intermolecular interactions. Physicochemicalcharacterizations carried out in this concentration range or obtained by extrapolation to infinite dilution
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3336 Langmuir, Vol. 9, No. 12, 1993
h
M
:
z c (
F
Y
-
1
0.01
I
I
I
0.1
1
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C, (g/dl) Figure 3. Intrinsic viscosity ([VI) of (0) PGA and ( 0 )PGA-Clz vs NaCl concentration (Ostwald-typecapillary viscometer).
therefore reflect the actual geometry (size, shape, ...I of the coil, only subordinate to the chemical composition of the macromolecule and its interaction with the solvent. The measurement of the intrinsic viscosity [ql, corresponding, for neutral polymers, to the extrapolation to infinite dilution of the Flory-Huggins20 equation qsp/c = [?I + kH[q12 c (where c is the polymer concentration and qsp the specific viscosity of the solution) is one of the most frequently used methods to obtain an accurate image of the expansion state. In addition, the Huggins coefficient k~ gives information on the solvation state, i.e. on the interaction between the coil and the considered solvent. Owing to the presence of charges, the reduced viscosity (qsp/c) of polyelectrolytes in pure water is not a linear plot of the concentration but generally varies as c-1/2.21 For this type of polymer, obtaining [ql requires performance of the viscosity measurements in the presence of screening salts, so as to mask the polyelectrolyte effect. Concerning PGA-Cl2 and its precursor PGA, the minimum salt concentration necessary for a linear plot qsp/c = f(c) is very low (0.01% NaC1). This is not surprising since the polyelectrolyte character of these two polymers is rather weak, owing to the high esterification ratio (over 70%)of the available carboxylic groups. Figure 3 shows the variation of [ql vs the saline concentration, for both PGA and PGA-Cl2. As can be anticipated from theory, [ql decreases for both polymers as the ionic strength is raised. Furthermore, [q]pGA-C12 < [q]pGA, whatever the salt concentration considered, In dilute aqueous solution, the dimension of an amphiphilic polyelectrolyte chain and, accordingly, its viscosity is the resultant of two opposing effects: the Coulombic repulsive forces between ionic charges borne by the macromolecule and the attractive interactions between ita hydrophobic segments. The effect of increasing ionic strength on PGA is limited to a progressive screening of the repulsions, leading, as expected,to a more and more compact coil. For PGA-CIZ, in superposition to this effect on repulsive charges, the ionic strength plays a favorable rolez2on the establishment or the reinforcement of hydrophobic interactions between (20) Huggins, M. L. J . Am. Chem. SOC.1942,64, 2716. R. M.; Cathers, G. I. J. Polym. Sci. 1949,4, 97. (21) FUOSE, (22) Tanford, C. The Hydrophobic Effect, 2nd ed.; J. Wiley: New York, 1980.
dodecyl groups. The consequence for PGA-Cl2 is that the expansion of its conformation, compared to that of PGA, is still more restricted and its viscosity further decreased. At last, since the higher the ionic strength, the stronger the hydrophobic effect, it is not surprising to observe that the difference, AIS], between the two macromolecules is increasingly large as the salt concentration is raised. The values of the Huggins coefficient are also significantly different for PGA and PGA-Cl2 in the whole range of ionic strength considered. For PGA, the values remain within the 0.3-0.5 range generally observed for nonassociating polymers.23 In opposition, the high values ( k >~ 1)obtained for PGA-Cl:! are a qualitative indication that polymer-polymer interactionsare more favored than those between the coil and the s o l ~ e n t This . ~ ~observation ~~~ is consistent with the hypothesis involvingthe formation of hydrophobic microdomains through intramolecular associations. In addition, the possibility of residual intermolecular interactions may as well be considered. At very low ionic strength (CS= 0.01%), A[?] is not equal to zero. This may correspond to some depolymerization during the introduction of dodecylamino groups in PGA. However, the molecularweights of both polymers, determined by SEC-LALLS, are not, within the accuracy limits of this technique, significantly different. An alternative hypothesis, involving the existence of hydrophobic microdomainsin solutions of very low ionic strength and even in pure water, can be proposed. Very similar results concerning the formation of concentration-dependent intra- or intermolecular hydrophobic associations have been recently obtained by Magny and ~ o - w o r k e r sby , ~viscometric ~~~~ study of sodium polyacrylate hydrophobically modified by introduction of increasing amounts of various long-chain alkylamines (Ca to c16). Pyrene is a hydrophobic probe with very low solubility in water. Its fluorescence spectrum at low concentration possesses fine structure whose relative peak intensities are highly sensitive to the polarity of the microenvironment.27 The ratio of the intensity of the highest energy vibrational band (11) to that of the third highest energy vibrational band ( 1 3 ) has proved to correlate with solvent polarity. In hydrocarbon solvents, the ratio11/13 is around 0.6. It is close to 1.1in ethanol and around 1.6 in ~ a t e r . ~ 7 This dependence of fluorescence fine structure on the microenvironment polarity has been used, for example, to investigate the formation of sodium dodecyl sulfate (SDS) micelles. Below the critical micellar concentration (cmc) the value of 11/13is close to that found in pure water. Above the cmc, the value of 11/13is 1.1,indicating that this probe is preferentially solubilized in the interior hydrophobic region of the micelle.28 Similarly, pyrene can be used to probe the formation of hydrophobic microdomains in microheterogeneous aqueous systems. I t is therefore well adapted to the study of PGA-Cl2 and appears as a complementary technique to viscometry. Figure 4 shows the variation of the 11/13value vs polymer concentration, in pure water, for PGA and PGA-Clz. (23) Riseman, J.; Ullman, R. J. Chem. Phys. 1951,19,678. (24) Moore, W. R.; Murphy, M. J.Polym. Sci. 1962,56, 519. (25) Magny, B.; Iliopoulos,I.;Audebert, R. Polym. Commun.1991,32, 456. (26) Magny, B. These de Doctorat, Universite de Paris VI, 1992. (27) Nakajima, A. J. Lumin. 1976, 11,429. (28) Kalyanasundaram,K.;Thomas, J. K.J. Am. Chem. SOC.1977,99, 2039.
Langmuir, Vol, 9, No. 12, 1993 3337
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. -7
3
m
I a,
1.25
I
1
I
I
0.001
0.01
0.1
1
-
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(W) Figure 4. Ratio of the intensity of fluorescencevibrational bands (Id&)of pyrene (c = 1.1 X 1o-S M)in the presence of (0)PGA and (0)PGA-CIz, vs polymer concentration, in pure water. The fluorescence spectrum of pyrene is hardly affected by the presence of PGA, in the whole concentration range tested. The value of 11/13stays above 1.8, indicating the aqueous nature of the environment probed. In contrast, in the presence of PGA-Cu and for polymer concentrations as low as 0.02%, 11/13 values continuously decrease to finally reach a plateau around 1.25, i.e. rather close to that experienced with SDS micelles above their cmc. This result is in agreement with the hypothesis proposed on the basis of viscometric data that hydrophobic microdomains already exist in pure water for PGA-Cl2. Considering the low amount of carboxylic groups available in ionized form on this polymer, it is not so surprising that the balance between electrostatic repulsions and hydrophobic attractionscan tend so readily toward the formation of such hydrophobic microdomains. The effect of increasing ionic strength on the variation of 11/13 vs PGA-Cl2 concentration is shown in Figure 5. One observes that the higher the ionic strength, the lower the polymer concentration necessary for the chain to start organizing in hydrophobic microdomains. Furthermore, the 11/13value at high polymer concentration (0.1 % and beyond) is lower as the salt concentration
1.2
I
0.001
0.01
0.1
c (g/dU Figure 5. Variation of ZllZa of pyrene (c = 1.1 X l V M ) in the presence of PGA-CIz vs polymer concentration, at various (0, 0 % ; P, 1 % ; 0 , 5 % ; 8 , 10% ) sodium chloride concentrations. is raised, indicating, as expected, that the environment probed by pyrene is increasingly hydrophobic with the ionic strength. The present preliminary work demonstrates that the introduction of C12 alkyl side chains on a derivative of sodium alginate produces peculiar physicochemicalproperties resulting from intra- or intermolecular hydrophobic associations, depending on the concentration range concerned. This may find extensions in many directions. Before approaching studies related to the gel state, one of our main concerns in the immediate future will be to investigate the physicochemical properties of derivatives, differing from PGA-Clz in both the substitution ratio and the length of the immobilized alkyl chain.
Acknowledgment. We wish to express our thanks to Dr. M. L. Viriot, DBpartement de Chimie Physique des RBactions, URA CNRS 328, Nancy, for her suggestions and help during the fluorescence measurements.
