In the study, chitosan (CS) was conjugated with trimethyl groups for the synthesis of N-trimethyl chitosan (TMC) polymers with different degrees of quaternization. Nanoparticles (NPs) self-assembled by the synthesized TMC and poly(gamma-glutamic acid) (gamma-PGA, TMC/gamma-PGA NPs) were prepared for oral delivery of insulin. The loading efficiency and loading content of insulin in TMC/gamma-PGA NPs were 73.8 +/- 2.9% and 23.5 +/- 2.1%, respectively. TMC/gamma-PGA NPs had superior stability in a broader pH range to CS/gamma-PGA NPs; the in vitro release profiles of insulin from both test NPs were significantly affected by their stability at distinct pH environments. At pH 7.0, CS/gamma-PGA NPs became disintegrated, resulting in a rapid release of insulin, which failed to provide an adequate retention of loaded insulin, while the cumulative amount of insulin released from TMC/gamma-PGA NPs was significantly reduced. At pH 7.4, TMC/gamma-PGA NPs were significantly swelled and a sustained release profile of insulin was observed. Confocal microscopy confirmed that TMC40/gamma-PGA NPs opened the tight junctions of Caco-2 cells to allow the transport of insulin along the paracellular pathway. Transepithelial-electrical-resistance measurements and transport studies implied that CS/gamma-PGA NPs can be effective as an insulin carrier only in a limited area of the intestinal lumen where the pH values are close to the p K a of CS. In contrast, TMC40/gamma-PGA NPs may be a suitable carrier for transmucosal delivery of insulin within the entire intestinal tract.