Synthesis and Rheological Properties of Responsive Thickeners

Biomacromolecules 2008, 9, 2419–2429

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Synthesis and Rheological Properties of Responsive Thickeners Based on Polysaccharide Architectures C. Karakasyan,† S. Lack,‡ F. Brunel,‡ P. Maingault,‡ and D. Hourdet*,† Physico-Chimie des Polyme`res et des Milieux Disperse´s, UMR 7615, UPMC-CNRS-ESPCI, 10 rue Vauquelin, 75005 Paris, France, and Laboratoires Brothier, Z.A. B.P. 26, 49590 Fontevraud L’Abbaye, France Received April 13, 2008; Revised Manuscript Received June 5, 2008

New thermothickening copolymers were synthesized by grafting responsive poly(ethylene oxide-co-propylene oxide) [PEPO] onto three different polysaccharide backbones: carboxymethylcellulose [CMC], alginate [ALG], and carboxylated dextran [DEX]. The coupling reaction between carboxylic groups of biopolymers and the terminal amine of PEPO was activated at low temperature (T < 10 °C) in water by using carbodiimide and N-hydroxysuccinimide. In these conditions it was shown that the formation of amide bonds strongly depends on the concentration of reactive groups, which is limited by the viscosity of the polymer sample. While a full conversion was obtained for the low molecular weight dextran, the efficiency of grafting remains low (between 30 to 40%) for CMC and alginate, which give a solution of high viscosity even at low concentration. When studied in the semidilute regime, all the copolymer solutions clearly exhibit thermothickening behavior with a large and reversible increase of viscosity upon heating. The association temperature and the gelation threshold were shown to depend on polymer concentration as it is expected from the phase diagram of PEPO precursor. Similarly, the influence of added salt on PEPO solubility in water has been used to control the self-assembling behavior of copolymer formulations. The relative comparison between the three copolymers reveals that the amplitude of the viscosity jump induced by heating mainly depends on the proportion of responsive material inside the macromolecular architecture rather than the dimensions of the main chain. The high increase of viscosity, which can reach several orders of magnitude between 20 °C and body temperature, clearly demonstrates the potentiality of these copolymers in biomedical applications like injectable gels for tissue engineering.

Introduction Contrary to most aqueous and organic-based formulations that generally follow the Arrhenius law and see their rheological properties decreasing with increasing temperature, there is a special class of macromolecular additives, called thermoassociating or thermothickening polymers, which provides the opposite behavior. This unusual property has been reported initially with cellulosic derivatives (methyl, hydroxypropyl, ethyl (hydroxyethyl)) and their mixtures with ionic surfactants.1–7 Afterward, mainly during the last two decades, the thermoassociating behavior has been broadened to synthetic block copolymers of poly(ethylene oxide) [PEO] and poly(propylene oxide) [PPO]8–10 and then explicitly rationalized with graft copolymers tailored with responsive side-chains showing a lower critical solution temperature (LCST) phase transition in water.11–16 Whereas all these copolymers have different chemical structures, their associating properties follow similar rules. At low temperature, water molecules initially reduce their entropy by forming ice-like structures around the nonpolar groups of the polymer chains. As the temperature is increased, the thermal motions progressively lead to a very unfavorable situation for the water shell, which is destabilized. This breakdown goes with the aggregation of nonpolar groups and induces the selfassembling of macromolecules. If there is a well-balanced equilibrium between responsive stickers and the water-soluble * To whom correspondence should be addressed. Tel.: +33 (0)1 40 79 46 43. Fax: +33 (0)1 40 79 46 40. E-mail: [email protected]. † UPMC-CNRS-ESPCI. ‡ Laboratoires Brothier.

counterpart, a physical network can be formed provided that the concentration is high enough to percolate through the whole volume. These responsive copolymers are of great interest as they provide technological solutions for complex fluids that require improved rheological properties above a given temperature. This situation typically concerns a wide range of industrial formulations like suspensions, coatings, cosmetics, and drilling fluids,17–19 but also biomedical applications such as drug release, viscosupplementation of biological fluids, and tissue engineering.20–23 If we focus more particularly on responsive thickeners working around body temperature, poly(N-isopropylacrylamide) [PNIPAM], poly(N-vinylcaprolactam) [PVCL], poly(vinylmethylether) [PVME], and block or random copolymers of ethylene oxide and propylene oxide [PEPO] are very good LCST candidates. Among them mainly PNIPAM and PEPO have been used in the preparation of grafted structures based on synthetic backbones like poly(sodium acrylate) [PAA],13–15 poly(acrylamide),24 poly(ethylene oxide),25 and other water-soluble copolymers.12 For medical applications, like regeneration of damaged tissues for instance, responsive hydrogels must have excellent bioabsorption properties, and, for that purpose, biopolymers are preferred over synthetic ones. Nevertheless, despite the existence of various thermoresponsive grafted copolymers based on artificial or natural macromolecules, most of them exhibit either phase separation or collapse properties upon heating rather than true thermothickening behavior. Besides methylcellulose, the only artificial derivatives that have been reported to give homogeneous

10.1021/bm800393s CCC: $40.75  2008 American Chemical Society Published on Web 08/02/2008

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Biomacromolecules, Vol. 9, No. 9, 2008

Karakasyan et al. Scheme 1. Carboxylation of Dextran

Table 1. Composition and Average Molar Mass of Polymer Precursors

polymer CMC ALG DEX PEPO

average number of monomer unitsa 200 200 150 (PO) ) 37/(EO) ) 7

Mn (g/mol) b

40000 40000b 25000b 2500c,d

[η] (mL/g) b

363 350b 15b

PI 40 5 1.9 1.05

a Intrinsic viscosities [η] were determined by SEC. Average number of monomer units and composition were obtained from Mn for homopolymers and 1H NMR for copolymers. b Number average molar masses (Mn) and polydispersity indexes (PI) were determined by SEC. c 1H NMR. d Mass spectrometry.

solutions with thermogelation properties were prepared on the basis of carboxymethylcellulose [CMC], chitosan [CHI], and hyaluronan [HYA]: CMC-g-PEO,12 CMC-g-PNIPAM,26,27 CHIg-PEO,22 CHI-g-PEPO,28 and HYA-g-PEPO.23 In this context, our main motivation was to develop new thermoassociating grafted biopolymers able to display sol/gel transition around body temperature. For that purpose, we have chosen to modify three different polysaccharides, CMC, alginate [ALG], and dextran [DEX], by grafting PEPO as responsive stickers. The choice of these biopolymers, currently used in biomedical applications, was guided by their good biocompatibility and the broad spectrum they offer in terms of composition, molar mass, and chain flexibility. Concerning the responsive stickers, PEPO was preferred over PNIPAM, as block or random copolymers of EO and PO are widely used in biomedical technologies like drug carriers or tissue regeneration. For instance, PEO-PPO block copolymers have been reported to be nonirritating when applied topically or subcutaneously to produce little irritation following intramuscular or intraperitoneal administration29 and to show good cytocompatibility when used in contact with different cell types.30 Even though PEPO derivatives are nondegradable, molecules with a molecular weight below 10-15 kg/mol are usually filtered by the kidney and cleared in urine.31 Among the very large PEPO family, we specially choose Jeffamine M-2005 from Texaco, which provides a convenient transition temperature for our purpose and has been successfully used to prepare thermothickening copolymers from very different architectures: PAA-g-PEPO,32,33 CHI-g-PEPO,22 and HYA-gPEPO.23 The synthesis of these new grafted copolymers and their self-assembling properties in semidilute aqueous solution are reported in this paper.

Experimental Section Reagents and Solvents. 1-[3-Dimethylaminopropyl]-3-ethylcarbodiimide hydrochloride (EDC, 98%, Acros), N-hydroxysuccinimide (NHS, 98%, Aldrich), succinic anhydride (SA, Aldrich), 4-dimethylaminopyridine (DMAP, 99%, Aldrich), triethylamine (NEt3, 99,5%, Aldrich), lithium chloride (LiCl, 98%, Fluka), and all organic solvents, including anhydrous dimethylformamide (DMF, 99.8%, Aldrich), ethanol (EtOH, 99%, SDS), and diethylether (Eth, 99.5%, SDS), were used as received. Water was purified with a Millipore system combining inverse osmosis membrane (Milli RO) and ion exchange resins (Milli Q). Polymer Precursors (see Table 1). Carboxymethylcellulose (CMC, Prolabo), with a degree of carboxymethylation DS ) 0.65 (obtained from titration), sodium alginate (ALG, G/M ) 0.6, Brothier), and Dextran T40 (DEX, Pharmacia) were received under powder form and used without purification.

Scheme 2. Grafting Reaction of PEPO-NH2 onto Alginate (Mannuronic Unit was Used to Symbolize Alginate)

Jeffamine M-2005, an amino terminated poly(ethylene oxide-copropylene oxide) (PEPO) with 84 mol % of propylene oxide, was kindly supplied by Huntsman (Belgium) and used as received. The average composition and molar mass of polymer precursors are reported in Table 1. Carboxylation of Dextran (τ ) 30%). The introduction of carboxylic groups on dextran backbone was carried out by reacting succinic anhydride onto hydroxyl functions in anhydrous conditions (see Scheme 1). A total of 5 g of dextran (31 mmol of monomer units) and 1.35 g of LiCl (31 mmol) were initially dried overnight in a three-necked flask at 100 °C under vacuum. Once dried and fitted with a reflux condenser, a CaCl2 guard tube, and a septum, 125 mL of anhydrous DMF was introduced into the vessel, and the mixture was heated at 80 °C under stirring and nitrogen bubbling until complete dissolution. Succinic anhydride (0.93 g, 9.3mmol) was then rapidly added, followed by 103.8 mg of DMAP (0.84 mmol) and finally by 102 µL of NEt3 (0.75 mmol). The reaction was left under a nitrogen atmosphere and continuous stirring during 16 h at 80 °C. At the end of the reaction, the main part of DMF was evaporated under reduced pressure, and the concentrated mixture was diluted in 150 mL of water. The polymer solution was then purified by dialysis against pure water (Spectra/Por membrane cutoff ) 6000-8000 daltons) and finally recovered by freeze-drying. The yield of succinated dextran after purification is about 60%. Grafting Reaction. The coupling reaction between carboxyl groups of polysaccharides and terminal amine of PEPO was carried out in cold water using EDC/NHS as coupling reagents (see Scheme 2). The same procedure has been followed for the three copolymers, except the concentration of the reaction medium, which has been modified to avoid a large viscosity. All the conditions of the synthesis are given in Table 2. A typical reaction can be summarized as follows. In a reaction vessel equipped with a magnetic stirrer, 5 g of polysaccharide was dissolved under stirring in 100 mL of water for at least 24 h at room temperature. A total of 5 g of PEPO (2 mmol of NH2) was separately dissolved in 50 mL of cold water to get a homogeneous solution and the pH was adjusted around 5-6 with hydrogen chloride (1 mol/L). After cooling, the polysaccharide solution in an iced bath at T ) 2-3 °C, the solution of PEPO was slowly added, and the pH was controlled again and adjusted to pH ) 5-6 if necessary. After 1 h of mixing under stirring, 0.23 g of NHS (2 mmol) and 1.22 g

Synthesis and Properties of Responsive Thickeners


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Table 2. Synthesis of Thermoresponsive Polysaccharides graft copolymers precursors polysaccharide (g) PEPO (g) NHS (g) EDC (g) H2O (g) weight fraction of PEPO in the copolymera grafting efficiency (%) Sb

ALG-gPEPO

CMC-gPEPO

DEX-gPEPO

5 5 0.23 1.22 160 0.28

5 5 0.23 1.22 230 0.25

5 5 0.23 1.22 160 0.5

37 6.2

33 5.3

100 10

a Weight fractions of PEPO are determined from 1H NMR analysis of the copolymer. b Average number of PEPO grafts per copolymer chain.

of EDC (6.4 mmol), dissolved separately in 5 mL of water, were slowly added into the reaction vessel, and the reaction was allowed to proceed over a period of 16 h below 10 °C. Then the copolymer was progressively precipitated under vigorous stirring in ethanol at room temperature. The precipitate was washed several times with ethanol to remove unreacted reagents and carbodiimide byproduct, filtered off, and finally dried under vacuum. In the case of dextran copolymer, the reaction medium was directly dialysed as previously described for carboxylated dextran using cold water during the two first exchanges. 1 H NMR. Polymer precursors and modified samples were characterized by 1H NMR in D2O with a Brucker Advance 400 MHz spectrometer. Acetonitrile was used as an external standard with ALG and CMC copolymers to calibrate the spectra. Size Exclusion Chromatography (SEC). SEC analyses were carried out on polysaccharide precursors using a Waters system equipped with three Shodex OH-pak columns equilibrated at T ) 35 °C in aqueous solution (LiNO3 0.5 mol/L). A dual detection “viscometry/refractometry” allows an absolute characterization of the molar masses after an initial calibration of the columns with PEO standards. Preparation of Copolymer Solutions. All the studies were performed with semidilute aqueous solutions, either in pure water or in salted media, adding potassium carbonate (K2CO3) or potassium sulfate (K2SO4). For each solution, copolymer, cosolute, and solvent were weighted, mixed, and allow to dissolve on a shaking table (250 rpm) until homogeneous solutions were obtained. Then the samples were left to equilibrate for two days at room temperature prior to analyses. The polymer concentrations are given in weight percentage (%) and the salt concentration is expressed in moles of salt per kg of solution (m). Rheology. The viscoelastic properties of graft copolymers in aqueous solutions were studied in the semidilute regime using a stress-controlled rheometer (TA Instruments AR1000) equipped with a cone/plate geometry (diameter 40 mm, angle 2°, truncature 55.9 µm). The experiments were performed in the linear viscoelastic regime, which was established for each sample by a stress sweep at the lowest frequency. The temperature was controlled by a high power Peltier system that provided fast and precise adjustment of the temperature during heating and cooling stages. The experimental conditions were fixed at constant frequency (1 Hz) and shear stress (between 0.02 and 5 Pa). Particular care was taken to avoid the drying of the sample by using a homemade cover that prevents water evaporation during the experiment. In these conditions, storage and elastic moduli as well as complex viscosity were recorded between 2 (or 20) and 60 °C. During heating and cooling, scans were performed at 2 °C · min-1 and the reproducibility was verified for each experiment. Differential Scanning Calorimetry. Phase transition of PEPO induced by heating was studied by differential scanning calorimetry (DSC) with a microDSCIII from Setaram. Solutions of approximately 0.8 g, equilibrated with a reference filled with the same weight of solvent, were submitted to temperature cycles between 5 and 70 °C with heating and cooling rates of 1 °C · min-1.

Figure 1. 1H NMR spectrum (D2O) and structure of DEX-g-PEPO.

Results and Discussion Synthesis and Characterization of Copolymers. Several methods are available for carboxylation of dextran and we use here the coupling reaction between anhydride and hydroxyl groups which was reported by Zalipsky et al.34 Contrary to other reagents, like monochloroacetic acid, which needs to be used in a large excess, succinic anhydride is very reactive and the carboxylation can be carried out in stoichiometric conditions until high conversion.35,36 In the present study, the reaction was performed in anhydrous conditions to get a 30% carboxylation rate, and this objective was perfectly reached, as confirmed by 1 H NMR. We have reported in Figure 1 the 1H NMR spectrum of dextran after carboxylation and PEPO grafting, as the assignment of the different signals remain quite clear, even after these two modifications. The methylene protons of the succinic residue can be easily distinguished at 2.4-2.6 ppm, while dextran protons at position 5 and 6 appear at 3.8-3.9 ppm and the methyl proton of PO appears around 1 ppm. All the other protons belonging to dextran (positions 2, 3, 4, and 6′) or to methylene and methyne groups of PEPO appear between 3.2 and 3.7 ppm, except for the anomeric proton of the glucose unit (position 1), which gives a peak at 4.9 ppm (not shown on the enlarged spectrum). Use of carbodiimide for the coupling reaction between amino groups and carboxyl functions is a standard method in peptide chemistry, which has been widely reported in the literature. More particularly, such a reaction has been typically applied for the modification of polymer chains bearing carboxylic groups with small molecules or macromolecules end-functionalized at one or both ends with primary amines.12,37,38 When the precursors are soluble in organic solvent, such a reaction can be easily performed at moderate temperature using organosoluble reagents like dicyclohexylcarbodiimide (DCCI). In these conditions, the amide formation between COOH and NH2 groups is rapid and quantitative even for a very small excess of DCCI.32,37 Of course, the same reaction can be carried out in aqueous media, but in that case, the formation of amide strongly depends on the conditions of the reaction. As reported by Nakajima and Ikada,39 the pH of the reaction and the possibility for carboxylic acids to form anhydride intermediate are important requirements for the formation of amide bonds. As a consequence, the pH is a compromise between successive reactions. In the first step, EDC can react with carboxyl groups in a relatively narrow low pH range such as 3.5-5 to form an amine-reactive intermediate. This O-acylisourea intermediate is not very stable, and N-hydroxysuccinimide (NHS) can be added

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with EDC to form a more stable activated ester.40,41 The low pH also favors the formation of anhydride intermediates, which play an important role in the amide formation.39 On the other side, the amide formation involves the reaction between previous intermediates (O-acylisourea, NHS-activated, or anhydride) and nonionized amines, which prevail at high pH. Obviously, the ionization equilibrium depends on the pKa of the amine but, generally, pH ) 5 is a good compromise between the abovementioned requirements. Moreover, noncyclizable carboxylic functions are known to be less reactive than cyclizable ones. For instance, carboxylic functions of poly(acrylic acid), which are able to form anhydride intermediates with carbodiimide, readily form the corresponding amides in the presence of amine groups.39 In the present case of polysaccharides, which are mainly soluble in water, the choice of aqueous medium for synthesis is natural, but we also have to consider the thermodynamic properties of PEPO in water. As a matter of fact, the range of water-solubility of PEPO is very narrow, and a temperature below T ) 15 °C is required to work in homogeneous conditions with PEPO concentrations lower than 10%. These conditions were fulfilled during our experiments and, accordingly, we expect to get a random distribution of PEPO side-chains along the polysaccharide backbones. The impact of graft solubility during synthesis on their distribution into the copolymer chain has been clearly identified in the case of hydrophobically modified water-soluble polymers.37,42 Similarly, we can extrapolate that for the coupling reaction performed above the LCST of PEPO, it will be possible to get a blocky distribution with different self-assembling behavior and rheological properties. From the experimental data reported in Table 2, we can see that the grafting reaction is not complete for polysaccharides and this result is quite unusual if we compare to the numerous grafting reactions that have been performed at higher temperature but in similar conditions with poly(acrylic acid) derivatives, either in organic solvent or in water.12,32 As discussed above, we must consider that carboxylic groups of polysaccharides are noncyclizable and that they cannot readily form anhydride intermediates. Nevertheless, the results obtained with carboxydextran refute this hypothesis, as the grafting is almost complete in this case. We can also take into account the accessibility of COOH, which is different between alginates (COOH being directly attached to the backbone) and CMC or succinylated dextran, which involve a spacer of varying size. Nevertheless, there is not a significant difference for the grafting efficiency between alginate and CMC. Another important experimental parameter is the relative concentration of reagents, but as the conditions are roughly the same for alginate and dextran modifications, it seems more realistic to take into account the viscosity of the reaction medium or more explicitly the degree of chain overlapping. This parameter can be roughly estimated byZ = C[η], where C is the concentration of polysaccharide during the modification step and [η] is its intrinsic viscosity in these conditions. For simplicity, the intrinsic viscosity determined by SEC in LiNO3, 0.5 mol/L, is used as reference (see Table 1). On this basis, we get very high values of overlapping for grafting reactions performed with ALG (Z ) 11) and CMC (Z ) 8). In these conditions, which correspond to the beginning of the entangled regime,43 a lower mobility and a weaker reactivity could be expected for the macromolecular reagents. By comparison, higher conversions were obtained, 52 and 80%, for coupling reactions performed with CMC at a lower degree of overlapping with PEO (Z ) 1.7)12 and PNIPA (Z ) 2.5).26 Similarly, a full conversion is obtained in the present work for

Karakasyan et al.

the modification of dextran performed just around its overlap concentration (Z ) 0.5). The problem of viscosity is especially acute in the present case because the reagents are not molecular, but macromolecular or at least oligomeric. Moreover, as the grafting of PEPO is performed at low temperature, it is important to maintain the reactivity of the macro-amine as high as possible compared to side-reactions that involve hydrolysis of the reaction intermediates. This is clearly the central problem that needs to reach a compromise: how to reduce the viscosity of the reaction medium without strongly decreasing the concentration of the reagents? An alternative would be to use large amounts of EDC/NHS to activate most of carboxylic acid units. This possibility has been reported by various authors,44–46 but it remains unsatisfactory from the point of view of future developments, especially if the reaction is intended just to modify a few percent of monomer units. Moreover, the introduction of large amounts of reactants (EDC/NHS) can produce ester bonds between the hydroxyl and the carboxyl groups of the polysaccharide chain. Such reaction has been used for instance by Zhang to prepare carboxymethyldextran/PNIPAM chemical gels.47 All these aspects are actually under investigation with the idea to extend the concept of thermoassociating polymers to a wider range of natural thickeners. Finally, the important conclusion concerning the structure of the copolymers is that they all contain a significant weight fraction of PEPO: between 25 and 50% (see Table 2). These fractions correspond to an average number of 5-10 responsive stickers per chain of polysaccharide, which is a reasonable value with the objective to develop associating networks through interchain associations.12,16 Phase Behavior of PEPO in Aqueous Solution. The PEPO precursor is a random copolymer of propylene oxide (PO) and ethylene oxide (EO) end-capped with an amino group. Due to its average composition (37 PO and 7 EO), an intermediate behavior between PPO and PEO, closer to PPO, is expected. Short chains of PPO are known to undergo a phase separation upon heating. This transition is related to the dehydration process of the polymer that occurs with a rearrangement of water molecules initially solvating the polymer.48 Dehydration is an endothermic process, as energy is needed for disrupting hydrogen bonds between water and ether oxygen. This positive enthalpy is thermodynamically unfavorable and it is, therefore, the entropic contribution that governs the phase separation process: the entropy increases once the water molecules leave the hydration shell around the polymer to move freely in the bulk. Thermodynamic data for various concentrations and degree of polymerization are reported in the literature.49,50 Generally, the transition temperatures and enthalpies depend on concentration and degree of polymerization but for low concentrations and low molar masses (M < 4000 g/mol) the transition enthalpy is about 8 kJ/mol of PO units. Compared to PPO, PEO is much more soluble in water and the phase separation behavior takes place at higher temperature, typically above 100 °C. Moreover, a significant amount of water remains in the concentrated PEO phase after phase separation, while PPO solutions mainly give rise to pure PPO phase above their LCST. An exhausting review on PEO is given in ref 51. As for PPO, and contrary to PEO, the PEPO copolymer phase transition is an endothermic process that can be analyzed by DSC. The thermogram of a 5% solution of uncharged amino PEPO (pH ) 11) is given in Figure 2. The phase separation starts at about 18 °C, where the first clouding is visually observed. This temperature will further be referred as the onset



Figure 2. Thermogram and corresponding integration of heatflow for a 5% PEPO-NH2 solution (pH ) 11). Ton denotes the onset temperature and Tmax denotes the temperature at the maximum of the endotherm. Enthalpy of transition, ∆H, is given in kJ per mol of PO units. Table 3. Enthalpy and Transition Temperatures of Aqueous Solutions of PEPO solvent water, pH ) 11 water, pH ) 1.6 [K2CO3] ) 0.2 m

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Figure 3. Variation of viscoelastic properties of CMC-g-PEPO in pure water (Cp ) 3%; f ) 1 Hz) upon heating (small symbols) and cooling (large symbols).

CPEPO (%) Ton (°C) Tmax (°C) ∆H (kJ/mol PO) 1 5 10 5 5

21 18 16 24 12

30 26 24 30 19

8 8.3 8.6 5.6 8.5

temperature, Ton. The maximum of the endotherm occurs at Tmax ) 26 °C and, above 45 °C, there is no further enthalpic change: two macroscopic phases (PEPO-rich and water-rich) are obtained after settling. The transition enthalpy ∆H, calculated by integrating heatflow with time (Figure 2), is given in joules per mole of PO units, assuming that EO units do not contribute energetically to the dehydration process at this temperature. The integration curve offers a good tool to depict the phase transition progress as it readily informs about the breadth and the magnitude of the heat effect. In the case of PEPO solutions at pH ) 11, a small decrease of the transition temperature is observed with increasing concentration (see Table 3), while the enthalpy remains almost constant: ∆H ) 8.3 ( 0.3 kJ/mol of PO units, which is comparable to ∆H = 8 kJ/mol of PO units referred for pure PPO.49,50 The thermodynamic behavior of PEPO chains can also be analyzed on the basis of the Gibbs-Helmholtz equation by plotting the logarithm of the concentration versus the inverse of the transition temperature

∆H ) R

[

∂ln(CPEPO) ∂(1 ⁄ Ton)

]

(1)

P

When applied to the small set of data given in Table 3 (pH ) 11), we get ∆H ) 330 kJ/mol of PEPO chain, which gives ∆H ) 8.7 kJ/mol of PO units, in good agreement with direct DSC measurements. Under acidic conditions (pH ) 1.6, adjusted with HCl), the ionic groups -NH3+ and Cl-, located at the end of the PEPO chains strongly disturb the thermodynamic behavior of the binary system and stabilize the phase separation at a lower scale. As a matter of fact, no macroscopic phase transition can be clearly observed, and the solutions remain clear or become only slightly opalescent upon heating. Nevertheless, the phase transition can be easily evidenced by DSC. The data reported in Table 3 show that, at low pH, the endotherm is shifted at higher temperature (Ton increases from 18 to 24 °C for C ) 5%), and the enthalpy of the aggregation process decreases from

8.3 to 5.6 kJ/mol of PO units. This comparison is evidence of the impact of ionic contribution that decreases the dehydration extent of PO units by increasing the interface between PEPO phase and water. As reported for PEO, PPO, and most of LCST polymers, the solubility of PEPO in water can be strongly modified in the presence of additives like salts, surfactants, and cosolvents. The addition of inorganic salts is known to generally decrease their solubility in aqueous media as they behave as competitors toward water molecules. Each salt is specific, depending on both anion and cation, and the magnitude of the salting-out effect is generally indexed according to the Hoffmeister series with reference to the effect of salts on the solubility of proteins in water. In the present study, we will use potassium carbonate and potassium sulfate, which are known to decrease efficiently and similarly the solubility of LCST polymers.52 As expected, the data reported in Table 3 show that the addition of potassium carbonate (0.2 m) into the PEPO solution decreases its solubility. The transition temperature falls from 18 to 12 °C, while the enthalpy remains unchanged, indicating that the salt does not strongly interfere in the dehydration process of PO units. Viscoelastic Properties of Modified Polysaccharides. CMC-g-PEPO. The rheological properties of semidilute solutions of CMC-g-PEPO were investigated as a function of temperature. A first example is given for a 3% polymer solution in Figure 3, where the variations of dynamic moduli (G′ and G′′) and complex viscosity (η*) have been followed in the course of heating and cooling. First of all, we will notice that, in our experimental conditions (f ) 1 Hz and heating and cooling rates ) 2 °C · min-1), the viscoelastic properties are perfectly reversible without noticeable hysteresis. This reversibility demonstrates that kinetic effects do not interfere on the self-assembling behavior. This has been already observed for a lot of thermoresponsive copolymers based on PNIPAM or PEO responsive stickers in the limit of low or intermediate molecular weights (typically below 10 kg/mol) and for moderate scanning rates.15 Independently of the choice of the parameter, the thermograms clearly display two temperature domains. At low temperature, where the PEPO side-chains are still miscible with water, the solution properties of the copolymer are very close to those expected for the CMC backbone: the solution is mainly viscous and G′′ prevails on G′. The copolymer solution is characterized by a low activation energy (Ea = 20 kJ/mol), close to the activation energy of water itself (Ewater ) a 16 kJ/mol). Above the transition temperature (Tas), PEPO side-

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Figure 4. Temperature dependence of the complex viscosity of CMCg-PEPO in water (1 Hz): Cp 1% (O), 2% (b), 3% (0), 4% (9), 5% (]). Table 4. Characteristic Temperatures of CMC-g-PEPO in Aqueous Solutions 0 0 0 0 0.1 0.2 0.3 0.4 K2CO3 (m) 0 Cp (%) 1 2 3 4 5 3 3 3 3 Tas (°C) 39.5 34.1 32.2 30 27.3 28.0 23.8 19.2 15.8 Tx (°C) 53 48.3 40.0 30.3 45.1 31.0 24.4 20.6 Tx/Tas 1.06 1.05 1.03 1.01 1.05 1.02 1.02 1.02

chains start to self-assemble in microdomains, which form gradually the physical junctions of the reversible network. The small difference between the upturns of G′ (Tas = 30 °C) and G′′ or η* (Tas = 32 °C) versus temperature is related to the stronger sensitivity of the elastic modulus toward self-assembling. In the experimental conditions (f ) 1 Hz), a crossover between G′ and G′′ is obtained at Tx ) 48 °C. The elastic modulus gains one decade between 20 and 60 °C, while the loss modulus increases about 2 times in the same temperature range. Around Tx, determined in Figure 3 at a given frequency (f ) 1 Hz), G′ and G′′ have very similar frequency dependence and this crossover is, therefore, a good approximation of the critical temperature of the sol-gel transition,53 as defined following the criterions proposed by Winters et al.54 As shown in Figure 4, the viscoelastic properties strongly depend on the copolymer concentration. At Cp ) 1% in pure water, the temperature dependence of the viscosity remains rather flat with a singularity around 40 °C, where a slight upturn is observed. For this solution, G′′ dominates G′ in the whole range of temperature explored. The thermothickening behavior appears more clearly at higher concentrations (Cp > 1%), where all the solutions display a sharp increase of viscosity above the association temperature and a prevailing elastic behavior at high temperature. By using the criterion proposed by Dobrynin et al.,43 that is, that solutions of polyelectrolytes and neutral polymers start to form entanglements at the same relative viscosity ηe (ηe = 50ηsolvent), we can verify that the semidilute entangled regime for CMC-g-PEPO in water typically starts above Cp ) 1%. The values of Tx and Tas, which are extrapolated, respectively, from the crossover of dynamic moduli and from the departure of the complex viscosity from the original Arrhenius behavior, decrease concomitantly with increasing polymer concentration (see Table 4), as it should take into account the phase diagram of PEPO. Nevertheless, there is a significant difference between the phase transition temperatures corresponding to the macrophase

Karakasyan et al.

Figure 5. Temperature dependence of the reduced viscosity of CMCg-PEPO aqueous solutions (f ) 1 Hz): Cp 1% (O), 2% (b), 3% (0), 4% (9), 5% (]).

separation of PEPO (see Table 3) and to the microphase separation of CMC-g-PEPO (see Table 4). One reason is that, contrary to the enthalpy, which increases linearly with the conversion of the phase separation process, a minimum number of connections between polymer chains are needed to provide a macroscopic deviation of the initial behavior. By applying the Gibbs-Helmotz, eq 1, to the data series obtained with CMCg-PEPO solutions (Table 4), we get a transition enthalpy ∆H = 2.9 kJ/mol of PO units, which is very low compared to the value obtained for the macrophase separation of PEPO precursor (∆H = 8.3 kJ/mol of PO units). This means that the dehydration process of PEPO grafts is not quantitative in the case of the copolymer and that part of the PO units remains hydrated even at high temperature. The higher value of Tas and the lower value of ∆H obtained for solutions of graft copolymers can be correlated to the high level of constraints undergone by PEPO grafts to self-assemble once grafted onto the CMC backbone. The main hindrance comes from electrostatic repulsions between polyelectrolyte backbones that exert opposite forces against PEPO association. To make a direct comparison between the solutions, we have * plotted in Figure 5 the relative complex viscosity (ηrel ) against the reduced temperature (T/Tas). For that purpose, the Andrade equation (η* ) A exp (Ea/RT)) was applied to the viscosity data obtained in the low temperature range (T < Tas) to extrapolate the complex viscosity without interaction at all temperatures * (ηdis (T)). In the Andrade equation, A is a constant, Ea is the activation energy for viscous flow, R is the gas constant, and T is the absolute temperature. The relative complex viscosity (η*rel) was calculated by dividing the experimental complex viscosity * η*(T) by ηdis (T) at each temperature. As shown in Figure 5, such representation provides an interesting picture of the net contribution of PEPO selfassembling in the construction of the physical network. As previously described at low concentration (Cp ) 1%), the thermothickening behavior is very weak and the gain in viscosity is only twice for T/Tas ) 1.07, that is, about 20 °C above the association temperature. At higher concentrations (Cp ) 2, 3, and 4%), which correspond to the entangled regime, the viscosity enhancement is more important and seems almost independent of the polymer concentration, with a gain of about one decade over 20 °C. This superposition, observed for Cp ) 2 and 4%, means that the polymer solutions follow similar scaling relations (here, η ∼ C4.5 p ) when they are compared at the same degree of association (T/Tas). Similar results have been



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reported experimentally by Candau and co-workers42 in the case of hydrophobically modified polyacrylamide and can be supported by the theoretical work on sticky reptation initially developed by Leibler et al.55 According to this theory, which applies for entangled solutions, the zero-shear viscosity (η0) can be described by the following equation

η0 ∼ C33⁄8N7⁄2[S]2τb(1 - 9 ⁄ p + 12 ⁄ p2)-1

(2)

where C is the polymer concentration, N is the average degree of polymerization of the chain, [S] ) S/N is the molar ratio of stickers with respect to N (grafting ratio), p is the fraction of associated stickers, and τb is the average lifetime of a sticker in an association, assuming that stickers self-assemble by pairs. According to this theory, the viscosity of associating polymer solutions mainly depends on the number and the nature of stickers (S, τb, and p), while the scaling dependence of η0 with concentration and molar mass remains very close to that reported for unmodified polymers. Consequently, by using the relative viscosity, as plotted in Figure 5, we effectively emphasize the net influence of the stickers

ηrel ∼ [S]2τb(1 - 9 ⁄ p + 12 ⁄ p2)-1 ) [S]2 · τb · F(p)

Figure 6. Temperature dependence of the complex viscosity of CMCg-PEPO in aqueous solutions: Cp ) 3%; f ) 1 Hz; [K2CO3] ) 0 m (O), 0.1 m (b), 0.2 m (0), 0.3 m (9), 0.4 m (]).

(3)

As [S] is constant for a given copolymer, eq 3 indicates that τb and p should be similar for a given degree of association for concentrations ranging between 2 and 4%. Contrary to conventional hydrophobically modified water-soluble polymers, which exhibit high viscosity at low temperature with a continuous thermothinning behavior, copolymers tailored with LCST sidechains, like PEPO, display the opposite trend due to the exothermicity of the demicellization process (phase separation being endothermic). As a matter of fact the disengagement process of PEPO from the micelle becomes more and more unfavorable upon heating and consequently the average duration of associations increases with temperature. If we assume that τb follows an Arrhenius behavior with temperature, it is possible to extrapolate in this concentration range negative energies, which are about -90 kJ/mol. Unexpectedly, the slope of the 5% solution is weaker than the previous ones. This effect can be understood by taking into account the frequency-dependence of the viscosity (η ∼ f-R, with R = 0.8), which is progressively shifted toward lower frequencies with increasing concentration and/or degree of association. Consequently, the viscosity determined at a fixed frequency (f ) 1 Hz) no longer remains Newtonian and it rapidly decreases compared to η0. By working at a fixed frequency, we have to consider that the concentration has two main effects on the thermothickening behavior. The first contribution is the increase of the connectivity above the percolation threshold that mainly increases the relaxation time of the network (τN ∼ τb · F(p)). This is typically observed by comparing the relative viscosity between 1 and 2% in Figure 5. The other consequence of the concentration is the nonNewtonian behavior, which starts to appear at frequencies higher than (τN)-1. By working at a fixed frequency, this softening effect is all the more important since the concentration increases and this particular point is clearly evidenced between 4 and 5%. As it is well-documented for PEO derivatives in aqueous solution, their solubility can be easily modified and controlled by adding co-solutes. For instance, the salting-out effect of potassium carbonate is shown in Figure 6 for a fixed copolymer concentration (Cp ) 3%). As previously described for PEPO grafts (see Table 3), the addition of K2CO3 lowers their solubility in water and decreases consequently the association temperature of CMC-g-PEPO (see Table 4). At the same time, the addition

Figure 7. Reduced viscosity of CMC-g-PEPO aqueous solutions versus reduced temperature. Cp ) 3%; f ) 1 Hz; [K2CO3] ) 0 m (O), 0.1 m (b), 0.2 m (0), 0.3 m (9), 0.4 m (]).

of salt partly screens the electrostatic repulsions between CMC backbones and makes the association easier and stronger. The net thermothickening effect due to increasing concentration of potassium carbonate is evidenced more clearly by plotting the data under reduced coordinates (see Figure 7). The negative energy extrapolated from the thermothickening process (slope of Ln(η) versus (1/T)) decreases progressively from -90 kJ/ mol of copolymer in pure water to -120 kJ/mol in [K2CO3] ) 0.4 m. So it appears that added salt readily decreases the association temperature of the copolymer but the impact on its viscosity enhancement remains rather weak. This point can be explained putting forward the low sensitivity to ionic strength of the semirigid backbones like CMC or alginates as it was reported earlier by Smidsrød and Haug.56 ALG-g-PEPO. Compared to CMC-g-PEPO, the solutions of ALG-g-PEPO are less viscous at room temperature and we have chosen to study more specifically this copolymer in [K2SO4] ) 0.2 m to enhance its thickening properties in the temperature range investigated. The dynamic properties of a 5% solution given in Figure 8 display a typical thermothickening profile, which is qualitatively very close to the one depicted for CMCg-PEPO in Figure 3. The association process starts at about Tas ) 17.7 °C and the crossover between G′ and G′′ takes place 20 °C above at Tx ) 37.2 °C.

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Figure 8. Variation of viscoelastic properties of ALG-g-PEPO in aqueous solution (Cp ) 5%; [K2SO4] ) 0.2 m; f ) 1 Hz) upon heating (small symbols) and cooling (large symbols).

Karakasyan et al.

Figure 10. Frequency dependence of the complex viscosity of ALGg-PEPO in aqueous solution (Cp ) 5%; [K2SO4] ) 0.2 m). T ) 10 °C (O), 20 °C (b), 35 °C (0), 40 °C (9), 45 °C (]), 50 °C ([).

Figure 11. (A) Temperature dependence of complex viscosity of ALGg-PEPO in [K2SO4] ) 0.2 m (1 Hz): Cp ) 1% (O), 2% (b), 3% (0), 4% (9), 5% (]). (B) Reduced plot. Figure 9. Frequency dependence of viscoelastic parameters (2G′, ∆G′′, bη*) of ALG-g-PEPO in aqueous solution at various temperatures (Cp ) 5%, [K2SO4] ) 0.2 m).

Complementary data are presented in Figure 9 where the frequency dependences of dynamic parameters are given at four different temperatures. Here the different steps of the sol/gel transition can be discussed more conveniently: (1) Below Tas (T ) 10 °C), the copolymer solution exhibits a Newtonian behavior in the whole frequency range (η0 = 1 Pa · s) with G′′ higher than G′. (2) Above Tas, the rheological properties dramatically increase and the viscosity is no longer Newtonian. At 40 °C, that is, close to the crossover temperature defined at f ) 1 Hz (Tx ) 37.2 °C), G′, and G′′ follow very close frequency dependences and this support very well the assumption that Tx is a good approximation of the critical gelation temperature. (3) Finally, at high temperature (T ) 50 °C), the associating system behaves as a gel with an elastic behavior prevailing on the whole frequency range explored. The frequency dependence of the complex viscosity is also plotted in Figure 10 for different temperatures to emphasize the strong increase of viscosity induced by PEPO self-assembling and the impact of the experimental conditions on the thickening assessment. For instance, one can extrapolate a

Table 5. Characteristic Temperatures of ALG-g-PEPO in Aqueous Solutions K2SO4 (m) Cp (%) Tas (°C) Tx (°C) Tx/Tas

0 5 24.6 ∼60 1.12

0.1 5 21.1 46.6 1.09

0.2 5 17.7 37.2 1.07

0.3 0.2 5 1 12.7 37.8 30.5 1.06

0.2 2 21.4 44.9 1.08

0.2 3 20.5 43.4 1.08

0.2 4 18.8 40.2 1.07

0.2 5 17.7 37.2 1.07

viscosity increase of three decades at ω ) 0.1 rad/s between 20 and 50 °C, but this large thermothickening is apparently reduced to only one decade when analyzed at higher frequency (ω ) 100 rad/s). As previously reported for CMC-g-PEPO solutions, the frequency dependence of the viscosity is responsible for the apparent weakening of the thermothickening process. This effect is clearly observed with solutions of ALG-g-PEPO in Figure 11, where the amplitude of thermothickening starts to decrease with increasing concentration above T/Tas = 1.06, that is, typically at temperature close to Tx (see Table 5). By applying the Gibbs-Helmotz, eq 1, to the data series between 2 and 5%, one can obtain the transition enthalpy of the self-assembling process in [K2SO4] ) 0.2 m: ∆H = 4.5 kJ/mol of PO units. This value is again lower than that determined with PEPO precursor (∆H = 8.3 kJ/mol of PO units) but higher than the enthalpy calculated for the same aggregation process in pure water with CMC-g-PEPO (∆H = 2.9 kJ/mol of


Figure 12. (A) Influence of salt on complex viscosity of ALG-g-PEPO aqueous solutions (Cp ) 5%; f ) 1 Hz): [K2SO4] ) 0 m (O), 0.1 m (b), 0.2 m (0), 0.3 m (9). (B) Reduced plot.

PO units). Screening effect of salt on the electrostatic repulsions between polyelectrolyte backbones is responsible for the increasing energy of the phase transition. This higher value can be correlated to a higher proportion of dehydrated PO, even if this process is far from being quantitative. When comparing the influence of polymer concentration in Figure 11, the rheological behavior at 1% is very peculiar because the concentration is not high enough to develop a tough gel at high temperature. Similarly, to the fact that a critical temperature of gelation (Tgel ≈ Tx) can be defined by working at a fixed concentration, it is also possible to determine a critical concentration threshold by working at high temperature. At T ) 60 °C, for instance, the concentration threshold should occur between 1 and 2%, either for CMC-g-PEPO in pure water (see Figure 4) or for ALG-g-PEPO in [K2SO4] ) 0.2 m (Figure 11). In the same way, by setting the copolymer concentration and the temperature, one can also find a critical salt concentration giving rise to the sol/gel transition. For example, in the case of a 5% aqueous solution of ALG-g-PEPO at T ) 50 °C, a critical potassium sulfate concentration should be obtained between 0 and 0.1 m (see Table 5 and Figure 12). From a general point of view, the reduced plot given in Figure 12 is very similar to the one depicted for CMC-g-PEPO in Figure 7. The slope of the thermothickening process slightly increases with increasing salt concentration and more particularly between 0 and 0.1 m for ALG-g-PEPO solutions. The energies, which can be extrapolated above Tas from the slope of Ln(η) versus (1/T), are similar to those obtained for CMCg-PEPO: -100 kJ/mol in pure water and -130 kJ/mol for [K2SO4] g 0.1 m. DEX-g-PEPO. The main problem with the dextran sample concerns its weak thickening ability that can be correlated to a high flexibility of the macromolecular structure combined to a low molar mass. As reported in Table 1, the intrinsic viscosity is very low ([η] ) 15 mL/g) and, consequently, high concentrations are needed to reach the semidilute regime: C* = 5%. For a better comparison with the two other copolymers we have chosen to work at a very high concentration (Cp ) 20%) in pure water. As shown in Figure 13, the viscosity of this solution determined at T ) 20 °C (η0 ) 63 mPa · s), that is, just before or very close to the beginning of the association process, is similar to those obtained with CMC-g-PEPO or ALG-g-PEPO at lower concentration, Cp ) 1 or 2%, respectively. Following the criterion of Dobrynin et al.,43 we can reasonably consider that at Cp ) 20%, dextran chains are entangled.


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Figure 13. Variation of viscoelastic properties of DEX-g-PEPO in pure water (Cp ) 20%; f ) 1 Hz) upon heating (small symbols) and cooling (large symbols).

Figure 14. (A) Influence of polymer architecture on thermothickening properties in water: (O) ALG-g-PEPO 5%, (b) CMC-g-PEPO 5%, (0) DEX-g-PEPO 20%. (B) Reduced plot.

The main characteristics of the solution of DEX-g-PEPO are the following: (1) A low temperature of association (Tas = 20 °C), which is related to the high copolymer concentration. Indeed, the relative PEPO concentration is about 10% in these conditions and Tas can be compared with the transition temperature of PEPO precursor determined at the same concentration: Ton ) 16 °C (see Table 3). As previously described, the difference between Tas and Ton emphasizes the influence of grafting and ionic interactions onto the thermodynamics of the association process of PEPO side-chains. (2) A high amplitude of thermothickening the complex viscosity increases 10000 times between 20 and 60 °C. To draw a comparison between the different copolymers we have plotted in Figure 14 the temperature dependence of the complex viscosity and its reduced form for three solutions prepared in pure water. As the concentrations, molar masses, and PEPO contents vary between the copolymer solutions, the reduced plot offers a more convenient form for the discussion. The first point is that CMC-g-PEPO and ALG-g-PEPO, which have equivalent weight fraction of PEPO and are studied in the same conditions (Cp ) 5% in water), also display very similar thermothickening signatures in the range T/Tas ) 1-1.06. At higher reduced temperatures, the non-Newtonian behavior of the solutions significantly alters the observation of their temperature dependence. Compared to these two copolymers which possess a low PEPO content, the thermoassociating properties of DEX-g-PEPO

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Karakasyan et al.

are dramatically different. From eq 3 we have seen that the relative viscosity (ηrel) of associating polymers in semidilute entangled solution was mainly independent of the size and the molecular weight of the copolymer and essentially mirrored the characteristics of the stickers: [S], τb, and p. As [S] is constant for a given architecture and independent of the temperature, it is interesting to draw a parallel between the temperature dependence of ηrel, that is, the dynamic properties of the solution and the thermodynamics of the phase transition. For instance, if we want to analyze the impact of the thermoassociation on the rheological properties, two key processes have to be considered. The first one concerns the self-assembling of PEPO chains, which involves an increasing number of stickers in the course of the phase transition. If we assume that at sufficiently high concentration most associations give rise to bridges rather than loops, we will simply consider the distribution between associating and pendant PEPO, which is taken into account in the conversion of the association process (p), as described by the rheological model (eq 3). This can be evaluated from the equilibrium constant of the association process (K) as follows

K)

1 e CPEPO

-∆H + T∆S ) exp( ( -∆G ) ∼ exp( -∆H RT ) RT RT )

) exp

(4) e CPEPO - CPEPO 1 p) )1CPEPO KCPEPO

(5)

withCPEPO, the total concentration of PEPO stickers, CePEPO, the concentration of free PEPO at equilibrium above Tas (pendant chains), ∆G, the standard free energy change for the transfer of one mole of PEPO from solution to the micellar phase, and ∆H and ∆S, the enthalpic and entropic contributions, respectively. From eqs 4 and 5 we can see that the conversion of PEPO from pendant chain to associated stickers is an increasing function of the temperature through the free energy of the phase transition. The second process which impacts the rheological behavior is related to the strength of the interactions between responsive stickers. As a matter of fact, the elementary step governing the dynamics of associations is the disengagement rate of the sticker 57,58 from the aggregate (β ∼ τ-1 During this event, the LCST b ). graft returns into the bulk solution for a while following a process that is comparable to the dissolution of PEPO in water, i.e. opposite to the coil/globule transition. Here the term globule is used to describe the conformation of PEPO chain inside the rich polymer phase (aggregate). As the mixing enthalpy of PEPO with water is exothermic, the exit of the PEPO graft from the aggregate becomes less and less favorable with increasing temperature and consequently the sticker will spend more and more time inside the aggregate. Under these considerations, we will simply assume that the lifetime of the sticker scales with the equilibrium constant of the association process and then with the free energy:

τb ∼ K ) exp

-∆H ∼ exp( ( -∆G ) RT RT )

(6)

A simple lecture of the above relations shows that the higher will be the temperature above the phase transition, the higher will be the number of sticky PEPO, the longer will be their lifetime in the aggregates and the higher will be the viscosity. Whatever is the temperature dependence of the viscosity, this discussion points out that thermoassociating polymers are resolutely different from conventional associating macromol-

ecules, like hydrophobically modified water-soluble polymers (HMWP). As a matter of fact, contrary to the solutions of HMWP, which become less viscous and elastic with increasing temperature,59 the viscoelastic properties of thermoassociating polymers are expected to increase not only during the formation of the physical network but also well beyond the association temperature in the so-called strong segregation regime. If we assume that the viscosity follows an Arrhenius dependence as proposed for the lifetime of the stickers in eq 6, the extrapolation of the experimental data plotted in Figure 14 between T/Tas ) 1-1.06 gives ∆H ) -(90-100) kJ/mol for CMC-g-PEPO and ALG-g-PEPO and ∆H ) -330 kJ/mol for DEX-g-PEPO. Although these values have to considered from a qualitative point of view, they clearly emphasize the large difference of association energy between the two set of polymers. At low content of PEPO, the stickers are rather far from each other and the association process involves weaker interactions between stickers embedded into smaller clusters as it was shown by neutron scattering with PAA-g-PEO derivatives.13 The large increase of viscosity previously reported between 1 and 2% in Figures 5 and 11 mainly corresponds to an abrupt change between loops and bridges in this concentration range. By comparison, the thermothickening behavior of DEXg-PEPO, which contains a double amount of stickers (50%), is much more important and similar to the one obtained with solutions of PAA-g-PEPO characterized by the same level of grafting.53 The proportion of responsive stickers appears clearly as a critical parameter for the design of responsive thickeners characterized by a large gap of their viscoelastic properties between low and high temperatures. From the experience we got on synthetic systems we can say that the thermodynamic and dynamic properties of the copolymer solutions are obviously related to the choice of the precursors: water-soluble and responsive chains. Nevertheless, a weight ratio 50/50 is generally a good compromise between the two contributions (watersolubility and association) to get a large and stable thermothickening effect without macrophase separation in water or salted solutions.12

Conclusion The main purpose of this paper was to transfer the technology and knowledge on thermoresponsive synthetic copolymers (chemistry and physico-chemistry) to biopolymers, which can find high potentialities in biomedical applications. Using PEPO as responsive sticker, we have shown that the “grafting onto” method based on the carbodiimide chemistry was an interesting way for the preparation of grafted architectures. Nevertheless, special attention has to be focused on each system to optimize the conditions of the reaction. The important points are the following: (1) The temperature of the reaction. It must stay below the cloud point of the LCST precursor to work in homogeneous conditions with a random introduction of the grafts. (2) The concentration of the reaction species. It should be as high as possible, but the reactions carried out with CMC and ALG have shown the limits of this rule. Consequently, the concentration of polysaccharides should remain at a reasonable level, well below the entangled concentration, in order that the viscosity does not interfere strongly on the kinetics of the reaction. The three copolymers prepared with PEPO have shown typical thermothickening behaviours with a sol/gel transition induced by heating. From a detailed analysis of the viscosity data using reduced coordinates, we have pointed out that the


temperature dependence of the viscosity could be split into two functions. The first one corresponds to the viscosity of the polysaccharide backbone at a given concentration. The second one is the net viscosity enhancement, which depends on various parameters: 1. Polymer Concentration. This parameter could be critical in the range of interchange loops to bridges corresponding to the sol/gel transition. In the semidilute entangled regime, where most stickers are assumed to self-associate intermolecularly, each copolymer has its own thermothickening signature. This later remains almost the same in this concentration regime as far as the frequency dependence of the viscosity remains not too high. 2. Ionic Strength. There is a significant difference between solutions prepared in pure water, where electrostatic repulsions are opposed to the PEPO/PEPO attractions, and those prepared with added salt. Moreover, added salt has a pronounced effect on the temperature of association which progressively decreases with increasing amounts. For salt concentrations higher than 0.1 m, the thermothickening profiles are almost the same and this allows a good extrapolation of the rheological properties on the basis of the initial behavior at low salt concentration. 3. Grafting Extent. As previously discussed, this parameter is obviously the most important, as it is responsible for the thermothickening signature of the copolymer. From a general point of view, the design of the copolymer has to take into account the water-soluble moiety and its responsive counterpart: 50/50 in weight is a good compromise. It is not possible to conclude clearly on the influence of the molecular weight of the polysaccharide on the thickening ability, as the copolymers have not the same PEPO content and that viscoelastic properties are strongly frequency dependent in our experimental conditions. Nevertheless, this peculiar point remains an important issue for the development of physical hydrogels. Finally, all the properties reported in this paper in the semidilute regime are worth being investigated in the dilute regime where responsive nanoassembling objects could be prepared. Such work is in progress and will be reported later. Acknowledgment. Financial support for this project by ANR PNANO is gratefully acknowledged.

References and Notes (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16)

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Synthesis and Rheological Properties of Responsive Thickeners

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