The use of non-viral delivery systems has numerous advantages in the area of gene therapy as this approach could circumvent the safety issues that may occur when using viral vectors. Hydrophobically modified chitosan containing five deoxycholic acid groups per 100 anhydroglucose units was synthesized to prepare self-aggregated nanoparticles in aqueous media and utilized to form complexes with plasmid DNA. Formation and various characteristics of DNA/chitosan self-aggregate complexes were investigated by electrophoresis, zeta potential measurement, and dynamic light scattering method. It was hypothesized that complex formation between DNA and chitosan self-aggregates can be strongly dependent on the charge ratio, pH, and incubation time. Migration of DNA on an agarose gel was completely retarded over a charge ratio (+/-) of 4/1 at pH 7.2, and the mean diameter of the complexes was determined to be 350 nm with unimodal size distribution (+/- =4/1; pH 7.2; incubation time, 30 min). The complex formation behavior was also regulated by pH of the media. The incubation time was unlikely to be the critical factor affecting the complex formation behavior due to the instant electrostatic interactions between negatively charged DNA and positively charged chitosan self-aggregates. This approach to controlling the characteristics of DNA/self-aggregate complexes could be critical in the design and tailoring of novel gene delivery systems using non-viral vectors. (c) 2005 Elsevier Ltd. All rights reserved.