Role of the pH on Hyaluronan Behavior in Aqueous Solution

Biomacromolecules 2005, 6, 61-67

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Role of the pH on Hyaluronan Behavior in Aqueous Solution Iuliana Gatej,†,‡ Marcel Popa,‡ and Marguerite Rinaudo*,† Centre de Recherches sur les Macromole´ cules Ve´ ge´ tales, CNRS, affiliated to Joseph Fourier University, BP 53, 38041 Grenoble Cedex 9, France, and Universite´ Technique “Gh.Asachi”, 67 Boulevard D.Mangeron, 6600 Iasi, Romania Received May 19, 2004; Revised Manuscript Received July 20, 2004

In this paper, we have examined the behavior of hyaluronan solutions at different pH values. A slight degradation is observed in acidic conditions (pH ) 1.6) and basic medium (pH ) 12.6) from molecular weight distribution analysis, but the rheological behavior is relatively not influenced much by the pH at the exclusion of two domains: around pH ) 2.5, a gel-like behavior is shown and is attributed to cooperative interchain interactions due to the reduction of the polymer net charge and may be the protonation of the acetamido groups; for pH > 12, the decrease of viscosity is mainly attributed to a reduction of the stiffness of the polymeric backbone in alkaline conditions due to the partial breakage of the H-bond network. Introduction Hyaluronan (also called hyaluronate, hyaluronic acid, or HA) was previously extracted from bovine vitreous humor, rooster combs, or umbilical cords; then it was very expensive and certainly associated with some proteins. Now the same polysaccharide was recognized to be produced by bacteria such as Streptococcus zooepidemicus on a large scale with a good yield and a large degree of purity. Then the price decreased, allowing the development of its applications, but its contribution depends on the conditions of use. The chemical structure of HA is represented as a linear polyelectrolyte based on β1-4-D-glucuronic acid and β1-3N-acetyl-D-glucosamine alternated in the repeat unit. The main uses of HA are ophthalmic surgery,1,2 arthritic treatment, and, more recently, cosmetics.3,4 The work developed for a few years in our laboratory concerned mainly the bacterial HA under the native form in neutral pH. Loosely cross-linked HA (named hylan) allowing better rheological performances and especially a gel-like behavior in a large range of frequencies is also produced for viscosupplementation in arthrosis treatment.5 This paper concerns the role of pH on the physicochemical properties of HA in aqueous solutions. Experimental Section HA is a bacterial sample produced by ARD Cy (Pomacle, France). It is prepared under the sodium salt form6 and characterized by steric exclusion chromatography (SEC) using a Waters Alliance GPCV2000 (U.S.A.) equipped with three detectors on-line: refractometric and viscometric detectors associated with a multiple-angle laser light scattering detector from Wyatt (U.S.A.).7 The concentration * Corresponding author. Tel.: 33476037627; 33476547203. E-mail: [email protected]. † CNRS. ‡ Universite ´ Technique “Gh.Asachi”.

injected is in the range of 0.5 g/L, and the volume injected is 108 µL on two columns in series (Shodex OH-pack 805 and 806). The eluent is 0.1 M NaNO3, and the temperature for elution is 30 °C; the weight-average molecular weight Mw and the polydispersity index I (I ) Mw/Mn) are given as characteristics of the polymers. The initial values are Mw ) 1.334 × 106 and I ) 1.49. For rheology, the HA solutions are prepared at a concentration of 10 g/L in 0.15 M NaCl; the pH was controlled by successive additions of HCl for the acidic medium and NaOH for basic conditions. The rheological behavior was studied using an AR 1000 rheometer from TA Instruments at 20 °C, when not precise. Plane-cone geometry is used with a 3.59° angle and 4-cm diameter. Dynamic experiments were performed in the linear domain at 5% deformation. The complex viscosity |η*| (Pa) is given at a low frequency corresponding to the Newtonian domain when it exists or at a fixed frequency (0.1 rad/s).8 The control of the structure of the polysaccharides can also be performed by 1H NMR (nuclear magnetic resonance) in the presence of a standard to calibrate the signal corresponding to the -CH3 of the N-acetylglucosamine unit. The standard generally used is 5 mM sodium succinate or dimethyl sulfoxide (DMSO) in D2O when the polymer concentration for NMR is around 5 mg/mL. But the NMR signals of specific groups, especially in 1H NMR, are quantitative only when they are mobile, that is, not involved in an ordered secondary structure such as a helical structure which can be stabilized by H bonds in stereoregular polymers or by specific interactions.9-11 1H NMR spectra were acquired on a Bruker AC300 spectrometer. Chemical shifts are given relative to external tetramethylsilane (TMS ) 0 ppm); the methyl signal from N-acetamido is at 1.93 ppm, and the -CH3 from DMSO is at 2.61 ppm. Results and Discussion A. Acidic Medium. The rheological behavior of the initial solution of HA is given in Figure 1. The initial pH of this

10.1021/bm040050m CCC: $30.25 © 2005 American Chemical Society Published on Web 11/06/2004

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Gatej et al.

Figure 1. Rheological behavior of initial HA at 10 g/L in 0.15 M NaCl at 20 °C and pH ) 6.05.

Figure 2. Complex viscosity as a function of the pH in acidic conditions. T ) 20 °C, polymer concentration Cp ) 10 g/L, and frequency is 1 rad/s.

solution is 6.05. Figure 1 is characteristic of a viscoelastic solution with a transition located at ω0 over which G′ becomes larger than G′′, meaning that entanglements exist giving an elastic character in this range of frequencies. The complex viscosity presents a plateau in the domain of low frequencies. In Figure 2, the complex viscosity is represented at a fixed frequency as a function of pH; at pH ) 2.42, the complex viscosity passes through a large maximum which is interesting to investigate. At this pH, the dynamic experiments demonstrate that G′ > G′′ in a large range of frequencies corresponding to a gel-like behavior. This process was previously described without any analysis (Figure 3).12-14 For pH values in the range of 2.86 up to 6.05 and at 1.6, the behavior in dynamic experiments remains similar just

modified by a slight change in the ionic concentration and a decrease of the net charge. Addition of NaOH in the pH ) 1.6 solution to increase the pH to 3.34 allows the initial behavior to nearly be recovered but at higher salt content. The complex viscosities for some solutions at different pH values are compared in Figure 4. This indicates that the mechanism of association assumed to be the cause of the large maximum in the range of pH ) 2.5 is reversible (comparison of pH ) 3.34 and 1.6) and that it corresponds to a critical balance of charges in the polymer; the reduction of the carboxylic group dissociation favors the H-bond formation (in relation with the intrinsic pK of this polyelectrolyte determined equal to 2.9 ( 0.1),15 but also the protonation of the -NH- group giving a positive net charge able to complex with the negative charge of few -COOH was suggested to interpret this mechanism even if the protonation constant for the acetamido group is not known at the time. Then the effect observed can be interpreted as due to an isoelectric point located around pH ) 2.5. From SEC experiments, it can be shown that after a short time at pH ) 1.6, the molar mass of the polymer decreases (one finds Mw ) 974 000 and I ) 1.59). In Table 1, the main rheological characteristics of the solutions are given for the different pH values tested. B. Characterization of the Gel-like Behavior. Dynamic experiments were performed as a function of temperature up to 50 °C at 1 Hz and pH ) 2.42. The results are given in Figure 5 where a characteristic temperature of ∼40 °C is shown to correspond to the transition from G′ > G′′ to G′′ > G′. From this temperature dependency, it comes that a gel-like structure stabilized by H bonds is formed when the -COO- dissociation decreases as mentioned in the previous paragraph. The cooperative interactions cause large thermosensitive interactions, as it was shown that the pH was

Role of the pH on HA Behavior in Solution

Biomacromolecules, Vol. 6, No. 1, 2005 63

Figure 3. Rheological behavior of HA at 10 g/L in 0.15 M NaCl at pH ) 2.42 and 20 °C showing a gel-like behavior in a large range of frequencies.

Figure 4. Complex viscosity of HA solution for different pH values: (O) 1.6; (b) 2.86; (0) 3.34; (9) 5.21; and (]) 6.05. Cp ) 10 g/L in 0.15 M NaCl at 20 °C.

not modified by temperature increase. From NMR of the acetamido CdO resonance, a large sharpening of the signal at a temperature larger than 40 °C due to the dissociation of H bonds between acetamido NH and carboxylate groups was previously demonstrated.11 In a second step, one investigates the role of polymer concentration on the gel-like behavior at pH ) 2.57. Addition

of solvent (0.15 M NaCl) at different contents allows the polymer concentration at a given pH to be varied; for these experiments, the systems were heated moderately after dilution and cooled for equilibration. A few data are given in Figure 6, where it is shown that with dilution a viscoelastic solution behavior is obtained down to 6.66 g/L in the frequency domain covered; specifically, a Newtonian plateau

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Gatej et al.

Figure 5. Evolution of the elastic modulus (G′) and viscous modulus (G′′) as a function of temperature for HA at pH ) 2.42. Conditions: HA solution Cp ) 10 g/L in 0.15 M NaCl at 1 rad/s.

Figure 6. Influence of the polymer concentration on the rheological behavior of solution. The G′ and G′′ are represented and show a rapid evolution of the viscoelastic behavior with polymer concentration. (O, b) 10 g/L; (], [) 6.66 g/L; (0, 9) 5 g/L.

exists only for the lower polymer concentration (e5 g/L). For concentrations of 5 g/L and lower, the viscosity drops suddently as indicated in Table 3.

Then one can conclude that when the pH of the solution is decreased by progressive addition of HCl, a thermosensitive gel is formed around pH ) 2.5 as a result of the



Table 1. Rheological Characteristics for HA in the Acidic Medium pH

G′a (Pa)

G′′a (Pa)

ω0 (rad/s)

|η*|b (Pa‚s)

6.05 5.61 5.21 4.38 3.34c 2.86 2.57 2.42 2.1 1.6

1.39 1.02 0.93 1.17 0.39 0.77 31.81 38.89 25.32 0.47

4.51 3.47 3.20 4.03 1.95 2.92 19.37 21.93 16.26 1.84

18.64 21.19 22.70 22.14 43.04 30.40 0.13 0.10 0.18 30.58

5.45 4.21 3.86 4.72 2.15 3.38 133 175 108 2.20

a Values taken at 1 rad/s. b Values taken at 0.1 rad/s. c Solution at pH ) 1.6 added with NaOH.

Figure 7. Complex viscosity as a function of the pH in basic conditions. T ) 20 °C, polymer concentration Cp ) 10 g/L, and frequency is 1 rad/s.

decrease of the carboxylate dissociation, favoring intermolecular interactions. A further decrease of the pH (see pH ) 1.6) causes the gel-sol transition; this transition may be related to the protonation of the acetamido groups causing an electrostatic repulsion between cationic polymers. As mentioned before, a balance of opposite charges is suggested as contributing to gelation also favored by the local stiffness of the HA chain.7,16,17 C. Basic Conditions. Progressive addition of NaOH on the HA initial solution shows that viscosity decreases slightly down to 11.58 followed by a transition in the range of pH ) 12 which corresponds to the pK of the -OH groups (Table 2; Figure 7). This transition was previously described by

Reed et al. who demonstrated a decrease of the radius of gyration of HA molecules without any change of the molar mass for progressive addition of NaOH.18 From Table 2, it is observed that the transition is nearly reversible; the values obtained for the different parameters for solutions at pH ) 11.5 and pH ) 11.58 are nearly identical, as well as the results for pH ) 3.82 (Table 2) and pH ) 3.34 (Table 1; Figure 8). Two hypotheses are able to justify a decrease of the viscosity at a pH larger than 12: first a decrease of the dimensions of the molecules based on a decrease of the stiffness and, second, a degradation of the backbone of the polymer with a decrease of the molecular weight. To investigate the mechanism involved in the quasi-reversible change in the viscosity at high pH, the SEC characterization of the different samples was performed. As shown in Figure 9, the molecular weight distribution is only slightly modified by addition of NaOH up to pH ) 12.6 followed by rapid reneutralization; Mw becomes Mw ) 1.183 × 106 and I ) 1.58 and are unable to justify the decrease of the viscosity

Figure 8. Complex viscosity of the HA solution for different pH values: ([) 12.33; (]) 12.6; (9) 11.5; (0) 10.95; (O) 6.15; and (b) 3.82. Cp ) 10 g/L in 0.15 M NaCl at 20 °C.


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Gatej et al.

Figure 9. Molar mass distributions in neutral conditions for initial pH (1; Mw ) 1.334 × 106; I ) 1.49), for HA having to be adjusted to pH ) 1.6 (2; Mw ) 9.74 × 105; I ) 1.59); and pH ) 12.6 (3; Mw ) 1.183 × 106; I ) 1.58) before reneutralization. Table 2. Rheological Characteristics for HA in Alkaline Conditions pH

G′ (Pa)

G′′ (Pa)

ω0 (rad/s)

|η*|a (Pa‚s)

6.15b

1.68 1.14 1.05 0.77 0.83 0.77 0.69 0.47 0.10 0.61 0.50

4.94 3.74 3.51 2.83 3.19 3.07 2.85 2.22 0.87 2.58 2.18

16.27 21.03 22.17 26.33 29.90 29.74 31.17 73.03

6.42 4.54 4.26 3.31 3.72 3.52 3.23 2.40 0.89 2.90 2.48

6.96 7.52 8.70 10.95 11.23 11.58 12.33 12.6 11.5c 3.82a

33.64 39.25

a Addition of HCl on the pH ) 12.33 solution. b Initial solution. c Addition of HCl on the pH ) 12.6.

Table 3. Influence of Polymer Concentration on the Gel-like Behavior pH

polymer concentration (g/L)

G′ (Pa) at 100 rad/s

ω0 (rad/s)

|η*| (Pa‚s) at 0.1 rad/s

2.57 2.7 2.34 2.6

10 6.66 5 2

102.5 21.35 4.02

0.13 3.74

133.5 6.25 0.73 0.033

observed; it is recalled that the rheology is nearly reversible. From these data, we are able to conclude that a reversible change in the stiffness of the HA molecule must occur when it passes through basic conditions at pH > 12.5. NMR experiments were developed to confirm this hypothesis; the mobility of the chain was tested from the relative height of NMR signals corresponding to the -CH3 group in the N-glucosamine unit in the presence of a wellcontrolled amount of DMSO used as a standard to calibrate the proton signals. In Figure 10, the 1H NMR spectrum taken at 80 °C is given showing the increases of chain mobility in the presence of sodium hydroxide (a) and when compared with the spectrum after reneutralization (b). In fact, the local mobility of the chain is related to the ratio of the integrals of the signal for the -CH3 protons at 1.9 ppm for HA and that of -CH3 at 2.6 ppm (for DMSO taken as the internal reference); this ratio passes from 0.91 in alkaline conditions (a) to 0.74 after reneutralization (b), respectively, indicating a larger mobility of HA molecules in the alkaline medium. In fact, for HA, some authors propose the existence of an

Figure 10. 1H NMR spectra for HA in D2O in the presence of DMSO as the reference (a) in the presence of NaOH pH > 12.5 and (b) after reneutralization.

ordered conformation in solution which is stabilized by H bonds.11,19,20 These bonds can be released in the presence of NaOH18 or urea.21 However, Sicinska et al.22 concluded from a detailed 1H and 13C NMR study of the repeating HA disaccharide that interactions with water predominated, and they found no evidence for long-lived intramolecular hydrogen bonds in aqueous solution for this disaccharide. On the opposite, on HA, the apparent intensity of the signal corresponding to the protons of the acetyl groups as well as those of the sugar units depends on the NaOH concentration



and on the temperature adopted for 1H NMR measurements. The exact conformation in aqueous solution is not yet established, but it may be possible that for the polymeric chain, dynamic H-bonded regions exist, controlling the average stiffness of the molecule. This hypothesis was proposed in the literature;23-25 then the fraction of H-bonded domains depends on the pH which would transfom the -OH groups in dissociated alcoholate (pK ∼ 12) at high pH, increasing the net charge of the HA and destabilizing the H-bond network.

(9)

(10)

(11) (12)

Conclusion (13)

The behavior of HA in aqueous solution depends on the pH; in this paper, the rheological behavior was examined and analyzed. It is shown that, in a large range of pH values, the rheological behavior remains nearly unchanged (2.8 < pH < 12); in the range of pH ) 2.5, a thermoreversible gellike behavior appears attributed to a cooperative interaction mechanism. A H-bond network may be formed when the net charge of the polymer decreases (reduction of the -COOH dissociation) and, maybe, when some protonation of the amido group occurs. At lower pH (pH ) 1.6), the polymer is resolubilized. This sol-gel transition is also pHreversible. A reversible conformational transition to a random coil is observed for pH > 12.5 due to the -OH groups dissociation in alkaline conditions; this mechanism reduces the number of H bonds between -OH and acetamido groups which control the local stiffness of the HA molecule.

(19)

References and Notes

(20)

(1) Hammer, M. E.; Burch, T. G. Viscous corneal protection by sodium hyaluronate chondroitin sulfate and methylcellulose. InVest. Ophthalmol. Visual. Sci. 1984, 25, 1329. (2) Balazs, E. A. The development of sodium hyaluronate as a viscosurgical material in ophthalmic surgery. In Ophthalmic Viscosurgery: A ReView of Standards, Techniques and Applications; Eisner, G., Ed.; Medicopea: Montreal, 1988. (3) Boudet, D.; Voskamp, K. L’acide hyaluronique: proprie´te´s et applications. Parfums, Cosmet., Aromes 1986, 68, 53. (4) Balazs, E. A.; Band, P. Hyaluronic acid: its structure and use. Cosmet. Toiletries 1984, 99, 65. (5) Balazs, E. A.; Leshchiver, E. U.S. Patent 4,582,865, 1984. (6) Rinaudo, M. Polysaccharide characterization in relation with some original properties. J. Appl. Polym. Sci., Appl. Polym. Symp. 1993, 52, 11. (7) Rinaudo, M.; Roure, I.; Milas, M. Use of steric exclusion chromatography to characterize hyaluronan, a semi-rigid polysaccharide. Int. J. Polym. Anal. Charact. 1999, 5, 277. (8) (a) Fouissac, E.; Milas, M.; Rinaudo, M. Shear-rate, concentration, molecular weight, and temperature viscosity dependences of hyaluronates, a wormlike polyelectrolyte. Macromolecules 1993, 26, 6945.

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(21) (22)

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(b) Milas, M.; Rinaudo, M.; Roure, I.; Al-Assaf, S.; Phillips, G. O.; Williams, P. A. Comparative rheological behavior of hyaluronan from bacterial and animal sources with cross-linked hyaluronan (hylan) in aqueous solution. Biopolymers 2001, 59, 191-204. Haxaire, K.; Buhler, E.; Milas, M.; Perez, S.; Rinaudo, M. Predictive and experimental behaviour hyaluronan in solution and solid state. In Hyaluronan: Chemical, Biochemical and Biological Aspects; Kennedy, J. F., Phillips, G. O., Williams, P. A., Hascall, V. C., Eds.; Woodhead Publ., Ltd.: Cambridge, 2002; Vol. 1, pp 37-46. Haxaire, K. Conformation du hyaluronane et interactions en solution et a` l’e´tat solide. Ph.D. Thesis, Grenoble University, Grenoble, France, 2000. Scott, J. E.; Heatley, F. Biomacromolecules 2002, 3, 547. Balazs, E. A. Sediment volume and viscoelastic behaviour of hyaluronic acid solutions. Fed. Proc. 1966, 25, 1817. Okamoto, A.; Miyoshi, T.; Abiocompatible gel of hyaluronan. In Hyaluronan: Chemical, Biochemical and Biological Aspects; Kennedy, J. F., Phillips, G. O., Williams, P. A., Hascall, V. C., Eds.; Woodhead Publ., Ltd.: Cambridge, 2002; Vol. 1, p 285. Milas, M.; Rinaudo, M. In Polysaccharides. Structural diVersity and functional Versatility, 2nd ed.; Dumitriu, S., Ed.; Marcel Dekker: New York, 2004 (in press). Beriaud, N.; Milas, M.; Rinaudo, M. In Polysaccharides. Structural diVersity and functional Versatility, 1st ed.; Dumitriu, S., Ed.; Marcel Dekker: New York, 1998; p 313. Haxaire, K.; Braccini, I.; Milas, M.; Rinaudo, M.; Perez, S. Conformational behavior of hyaluronan in relation to its physical properties as probed by molecular modelling. Glycobiology 2000, 10, 587. Fouissac, E.; Milas, M.; Rinaudo, M.; Borsali, R. Influence of the ionic strength on the dimensions of sodium hyaluronate. Macromolecules 1992, 25, 5613. Gosh, S.; Kobal, I.; Zanette, D.; Reed, W. F. Conformational contraction and hydrolysis of hyaluronate in sodium hydroxide solution. Macromolecules 1993, 26, 4685. Heatley, F.; Scott, J. E. A water molecule participates in the secondary structure of hyaluronan. Biochem. J. 1980, 254, 489. Scott, J. E.; Heatley, R.; Hull, W. E. Secondary structure of hyaluronate in solution. A 1H NMR. investigation in dimethyl sulfoxide solution. Biochem. J. 1984, 220, 197. Hirano, S.; Kondo, S. Molecular conformational transition of hyaluronic acid in solution. J. Biochem. 1973, 74, 861. Sicinska, W.; Adams, B.; Lerner, L. A detailed ′H and ′′C n.m.r. study of a repeating disaccharide of hyaluronan: The effects of temperature and counterion type. Carbohydr. Res. 1993, 242, 29. Almond, A.; Brass, A.; Sheehan, J. K. Oligosaccharides as Model Systems for Understanding Water-Biopolymer Interaction: Hydrated Dynamics of a Hyaluronan Decamer. J. Phys. Chem. B 2000, 104, 5634. Almond, A.; Brass, A.; Sheehan, J. K. Dynamic exchange between stabilized conformations predicted for hyaluronan tetrasaccharides: comparison of molecular dynamics simulations with available NMR data. Glycobiology 1998, 8, 973. Almond, A.; Sheehan, J. K.; Brass, A. Molecular dynamics simulations of the two disaccharides of hyaluronan in aqueous solution. Glycobiology 1997, 7, 597.

BM040050M

Role of the pH on Hyaluronan Behavior in Aqueous Solution

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