Role of the pH on Hyaluronan Behavior in Aqueous Solution Iuliana Gatej, ²,‡ Marcel Popa, ‡ and Marguerite Rinaudo* ,² 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 1-4-D-glucuronic acid and 1-3- N-acetyl-D-glucosamine alternated in the repeat unit. The main uses of HA are ophthalmic surgery, 1,2 arthritic treat- ment, 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 viscosupplemen- tation 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 form 6 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 scat- tering detector from Wyatt (U.S.A.). 7 The concentration 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 NaNO 3 , and the temperature for elution is 30 °C; the weight-average molecular weight M w and the polydispersity index I (I ) M w /M n ) are given as characteristics of the polymers. The initial values are M w ) 1.334 × 10 6 and I ) 1.49. For rheology, the HA solutions are prepared at a concen- tration 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 1 H NMR (nuclear magnetic resonance) in the presence of a standard to calibrate the signal corre- sponding to the -CH 3 of the N-acetylglucosamine unit. The standard generally used is 5 mM sodium succinate or dimethyl sulfoxide (DMSO) in D 2 O when the polymer concentration for NMR is around 5 mg/mL. But the NMR signals of specific groups, especially in 1 H 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 1 H 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 -CH 3 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 * Corresponding author. Tel.: 33476037627; 33476547203. E-mail: marguerite.rinaudo@cermav.cnrs.fr. ² CNRS. ‡ Universite ´ Technique “Gh.Asachi”. 61 Biomacromolecules 2005, 6, 61-67 10.1021/bm040050m CCC: $30.25 © 2005 American Chemical Society Published on Web 11/06/2004