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Electrostatic properties of three chitosans with fractions of N-acetylated units (F(A)) of 0.01, 0.13 and 0.49 were examined by electrophoretic light-scattering technique (ELS) and (1)H NMR spectroscopy. From the dependency of mean electrophoretic mobilities on pH, the pK(a) values were calculated. Despite their large differences in chemical composition, all chitosans had similar pK(a) values of 6.5-6.6. All chitosans also showed the same polyelectrolyte behavior when apparent pK(a) values were calculated according to Katchalsky and plotted as a function of the degree of ionization alpha. The intrinsic pK(a) values (pK(0)) extrapolated to zero charge were about 9. The results derived from an independent (1)H NMR study of the same chitosan samples showed no effect of F(A) on titration behavior of chitosan, confirming the results obtained by ELS.

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Hyaluronic acid (HA) was hydrolyzed using varying temperatures (40, 60, and 80 degrees C) and acid concentrations (0.0010, 0.010, 0.10, 0.50, 1.0, and 2.0 M HCl). The degradation process was monitored by determination of weight average molecular weight ( M w) by size-exclusion chromatography with online multiangle laser light scattering, refractive index, and intrinsic viscosity detectors (SEC-MALLS-RI-visc) on samples taken out continuously during the hydrolysis. SEC-MALLS-RI-visc showed that the degradation gave narrow molecular weight distributions with polydispersity indexes ( M w/ M n) of 1.3-1.7. Kinetic plots of 1/ M w versus time gave linear plots showing that acid hydrolysis of HA is a random process and that it follows a first order kinetics. For hydrolysis in HCl at 60 and 80 degrees C, it was shown that the kinetic rate constant ( k h) for the degradation depended linearly on the acid concentration. Further, the dependence of temperature on the hydrolysis in 0.1 M HCl was found to give a linear Arrhenius plot (ln k h vs 1/ T), with an activation energy ( E a) of 137 kJ/mol and Arrhenius constant ( A) of 7.86 x 10 (15) h (-1). (1)H NMR spectroscopy was used to characterize the product of extensive hydrolysis (48 h at 60 degrees C in 0.1 M HCl). No indication of de- N-acetylation of the N-acetyl glucosamine (GlcNAc) units or other byproducts were seen. Additionally, a low molecular weight HA was hydrolyzed in 0.1 M DCl for 4 h at 80 degrees C. It was shown that it was primarily the beta-(1-->4)-linkage between GlcNAc and glucuronic acid (GlcA) that was cleaved during hydrolysis at pH < p K a,GlcA. The dependence of the hydrolysis rate constant was further studied as a function of pH between -0.3 and 5. The degradation was found to be random (linear kinetic plots) over the entire pH range studied. Further, the kinetic rate constant was found to depend linearly on pH in the region -0.3 to 3. Above this pH (around the p K a of HA), the kinetic constant decreased more slowly, probably due to either a change in polymer conformation or due to an increased affinity for protons due to the polymer becoming charged as the GlcA units dissociated.

The "grafting onto" strategy was used to conjugate DL-lactic acid oligomers (OLA) to hyaluronan (HA) for the sake of developing novel degradable HA-based self-assembling polymeric systems. Grafting was achieved by reacting COCl-terminated OLA with cetyltrimethylammonium hyaluronate (CTA-HA) in dimethyl sulfoxide (DMSO). The resulting CTA-HAOLA conjugates were purified and turned to sodium form (Na-HAOLA) by dissolution in a phosphate buffer-DMSO mixture and successive dialyses against DMSO, ethanol, and water. In contrast, when the same protocol was applied to CTA-HAOLA, phase separation with gel formation was observed. The solution phase was composed of Na-HAOLA whereas the gel phase was made of mixed CTA-Na-HAOLA salt with ca. 25% of the carboxyl groups neutralized by CTA. Gelation was assigned to intramolecular hydrophobic associations between OLA and cetyl alkyl chains that complemented electrostatic interactions between CTA and HA COO- groups synergistically. Therefore, the corresponding stabilized CTA ions required more drastic conditions to be released. Under the selected dialysis conditions, the CTA-Na-HAOLA gels formed tiny tubes. Na-HAOLA and CTA-Na-HAOLA were characterized by FTIR, one-dimensional 1H and two-dimensional 1H NMR. The extent of grafting was ca. 5% per disaccharidic repeating unit, regardless of the molecular weight, as determined by NMR and capillary zone electrophoresis. Amphiphilic Na-HAOLA molecules were aggregated and formed spherical species in water according to size exclusion chromatography combined with multiangle laser light scattering detection. The critical aggregation concentration ranged between 0.2 and 0.35% (w/v), depending of the molecular weight of the parent hyaluronan.

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