Dielectric relaxation in Hyaluronic acid sodium salt

1. Dielectric relaxation in Hyaluronic acid sodium salt S.Dolanski Babić1,2, T.Ivek1, T.Vuletić1, S.Tomić1 and R.Podgornik3,4 1Institut za fiziku, Zagreb, Hrvatska 2Zavod za fiziku i biofiziku, Medicinski fakultet, Zagreb, Hrvatska 3Department of Physics, University of Ljubljana, Ljubljana, Slovenia 4J.Stefan Institute, Ljubljana, Slovenia

2. I Introduction II Samples III Low freq. Dielectric Spectroscopy IV Results V Plans

3. glucose Introduction - Hyaluronan (hyaluronic acid or hyaluronate) is simple polymer of disaccharides themselves - composed of glucuronic acid and N-acetylglucosamine - linked together via alternating glycosidic bonds - Molecular weight of one monomer is 400 Da

4. HYALURONAN IS- - found in all tissues and body fluids of vertebrates as well as in some bacteria - synthesized in the cellular plasma membrane - a major component of the synovial fluid and was found to increase the viscosity of the fluid - a major constituent in the vitreous of the human eye • an important component of articular cartilage, where it is present as a coat around each cell - is also a major component of skin, where it is involved in tissue repair ~1 nm Introduction Hyaluronan (HA) can be 25.000 monomers in length

5. Hyaluronic acid Sodium salt from Streptococcus equi sp. (BioChemika Fluka) - molecular weights of HA* are in the range 300 kDa- 1900 kDa - polydisperse HA fragments in the range 750- 5000 monomers (0.75 -5 mm) - pure water solutions in the range of 0.01-5 mg/mL - pure water: MilliPore, Milli-Q, 0.056 mS/cm Samples * K.Tamoto, Semin.Arthritis Rheum., Vol. 22(1993)

6. Impedance analyzerAgilent 4294measures capacitance, C and conductance, G (real part) Y(w)= G(w)+iwC(w) • Chamber for liquid samples – water solutions • conductivity: 1.5-2000 mS/cm • drops vol.: 50-200 mL • reproducibility 0.8% • long term (2 h) 2% Dielectric spectroscopy (40 Hz-100 MHz) Temperature control unit Temp. range:5°C - 60°C Stability:±0.1 K Pt

7. Y(w)=G(w)+iwC(w) • G(w) and C(w) of DNA solutions are measured • - These are subtracted for (G, C) of background (reference) NaCl solution with matching conductivity (at 100kHz) From complex conductance to complex dielectric function - This procedure enables to eliminate the electrode polarization effects, as well as other stray impedance effects.

8. (G-GNaCl, C-CNaCl) e(w)= e’(w)-ie’’(w) From complex conductance to complex dielectric function FITS to a sum of two generalized Debye functions - relaxation process strength, - 0, central relaxation time - symmetric broadening of the relaxation time distribution,1 - 

9. HA pure water solutions dielectric response spectra feature two modes (as DNA solutions) - two relaxation modes in kHz and MHz ranges Results

10. HF mode: De 4, 1-a 0.82 LF mode: De 60 for c < 0.2 mg/mL De 10 for c > 0.4 mg/mL 1-a 0.75 Results tLF tHF

11. Results semidilute regime above c*=0.001 mg/mL

12. a=0.5 a=0.4 Results

13. fHF - fraction of counterions participating in the HF process aHF- polarizability Results

14. Repeat the dielectric spectroscopy for Hyaluronic acid pure water solutions in the same range of HA concentration as reported here. • 2. Hyaluronic acid in NaCl electrolyte (with added salt of fixed or varying concentration), similar range of HA concentration as reported here. Plans