Rheological Studies of Hyaluronan/Modified Hyaluronan Mixtures and the Structure of Hyaluronic Solutions

The structure of solutions of hyaluronan (HA) and HA/HMHA (hydrophobically modified HA) mixtures were studied by means of shear viscosity measurements, oscillation, creep and relaxational tests. Influence of molar mass, concentration, the presence of salt and the HA/HMHA weight ratio is discussed. INTRODUCTION Hyaluronic acid is a naturally abundant linear polysaccharide that consists of repeating disaccharide structure units of β(1→3) linked D-glucuronic acid and Nacetyl-D-glucosamine. The disaccharide units are linked via β(1→4) glycosidic bond. HA contains negatively charged carboxylate groups, thus it is a polyelectrolyte and forms a salt form of hyaluronate or hyaluronan at physiological pH. HA occurs in extracellular matrix of vertebrates connective tissues with a wide range of molar masses from several hundreds to 10 millions g/mol. HA plays many pivotal functions in the organism such as a spacefiller or a scaffold for other macromolecules, creating a network with proteoglycans and proteins. HA, thus maintains viscoelasticity of the tissues due to its extraordinary rheological properties. HA also participates in various receptormediated cell processes 5, , e.g. mitosis, cell migration, tumor development, inflammation, etc. HA has found numbers of important applications in medicine, e.g. ophthalmology, visco-supplementary, osteoarthritis treatment, drug delivery 2, 7, , pharmacy and cosmetics due to its rheological and other unique properties. During technology processes, HA undergoes various mechanical stresses, thus rheology properties play important role in the processes and many applications of HA. Therefore, knowledge of a relationship between rheological parameters and structure of hyaluronic solutions is very helpful during these operations and utilizations. Previous studies on rheology of HA solutions were mostly based on rotational shear viscosity measurements and/or oscillatory measurements . In this paper we tried to investigate the structure of HA solutions not only using these techniques but also by submitting HA solutions to a sudden load of various intensities and duration observing rheological parameters during and after removing the load. Thus, we used relaxation, creep and recovery tests for probing breakdown and buildup the structures of HA solutions. Rheological Studies of Hyaluronan/Modified Hyaluronan Mixtures and the Structure of Hyaluronic Solutions Martin Chytil , Miloslav Pekař Institute of Physical and Applied Chemistry, Faculty of Chemistry, Brno University of Technology, Purkyňova 118, 612 00, Brno CPN, ltd., 561 02, Dolní Dobrouč 401 MATERIALS AND METHODS Materials Bacterially produced HA of two molar masses of 1.63, 1.69×10 g.mol and 5.6×10 g.mol, and octyl chain bearing hydrophobically modified HA (HMHA) marked as C8nhHA9 of molar mass of 4.5×10 g.mol and the degree of substitution (DS) of 100% were studied. HA was modified with a chemical linking of the alkyl chain via carbamate bond as described here. DS here corresponds to a number of the disaccharide units in percentage occupied with one alkyl group. The samples were produced in the CPN, ltd. Company. Methods Measurements were performed on rheometer RS Haake 100 with the cone-andplate (CP) geometry of the cone diameter of 60 mm and 1° cone angle, and on STRESSTECH Rheologica Instruments rheometer with the CP geometry of 40 mm/4° (NTNU, Trondheim). The oscillatory measurements were performed mostly in the range of frequencies, f = 0.005 – 10 Hz, at the strain value determined from the strain sweep measurements. Shear viscosity was measured using the up-anddown stress or shear rate scans. For investigation of the breakdown and buildup the solution structure a sudden impulse of shear rate or shear stress was applied to the sample and rheological respond was observed in time. All the measurements were performed at (25 ± 0.2) °C. RESULTS AND DISCUSSIONS Rheology of HA and HMHA solutions Previous rheology studies on HA have reported about non-Newtonian shearthinning behavior of HA aqueous solutions 3, 8 which is very advantageous in many applications. HA also displays extraordinary rheology behavior under various conditions and forms associated structures resulting e.g. in high viscoelasticity of the HA solutions, particularly of high molecular HA, by increasing the storage modulus, G’, (Fig.1). G’ increases with the concentration and molar mass of HA with the shift of the cross point (G’ = G’’) to lower frequencies. 1,E-03 1,E-01 1,E+01 1,E-03 1,E-01 1,E+01 frequency, f (Hz) G ', G '' (P a) 1,E-01 1,E+00 1,E+01 co m pl ex v is co si ty , η * ( Pa .s)