Polyelectrolyte complexes (PEC) formed from chitosan derivatives and insulin was prepared and parameters influencing complex formation were characterized. Turbidimetric titration, in combination with dynamic light scattering (DLS) and laser doppler anemometry (LDA), were used to study the complexation process. The morphology of the PECs was determined using atomic force microscopy (AFM). PEC formation was predominantly pH-dependent. Complexation with insulin occurred only above critical pH value (pHc) of 6.0 for all the chitosan derivatives investigated. Soluble PECs in the size range of 200-500 nm with spherical or subspherical morphology and smooth surface structure were obtained at optimized polymer/insulin charge ratios. Optimal conditions were obtained when the pH of PECs was in the range of 6.5-8.0, depending on polymer structure. The stability of PECs was influenced by polymer chain length. Only when the MW of the polymers was > or =25-kDa PEC precipitation could be avoided. An increase in the ionic strength of the medium accelerated complex dissociation. Chitosan methylation and PEGylation significantly improved the stability of insulin in the PECs. Moreover, the PEC could protect insulin from degradation even at 50 degrees C for at least 6 h. All complexes could be lyophilized without influencing the particle size, complex concentration, and stability of insulin. On the basis of our results, we suggest that interactions involved in PEC formation were predominantly electrostatic in nature, involving the positively charged amino groups of chitosan and the negatively charged insulin above its isoelectric point. Intranasal absorption of the polyelectrolyte nanocomplexes will be studied in vivo.