The purpose of this study was to prepare and characterize nanocarrier systems, which allow the application of pDNA vaccines and adjuvants to mucosal vaccination. Chitosan from a vegetal source (Agaricus bisporus) and of GMP quality was used to synthesize the derivative 6-O-carboxymethyl-N,N,N-trimethylchitosan (CM-TMC). Toll-like receptor-2 (TLR-2) agonist, Pam(3)Cys, was synthesized and coupled to CM-TMC through a polyethylene glycol (PEG) spacer. Successively, Pam(3)Cys decorated nanocarriers were prepared by complexation with plasmid DNA (pDNA) expressing green fluorescence protein (GFP), and characterized with respect to their physicochemical properties and protection of the included plasmid against DNase I enzymatic degradation. In vitro studies using phorbol 12-myristyl 13-acetate (PMA) stimulated macrophage-like THP-1 (mTHP-1) cells were focused on cytotoxicity of both polymers and particles, and their potential to stimulate IL-8 release via the TLR-2 pathway. Our results showed that the TLR-2 functionalized pDNA nanocarriers have the ability to complex and to protect pDNA against enzymatic degradation. pDNA nanocarriers were of around 400 nm in size, and displayed a positive zeta potential of 27.9 +/- 1.6 mV. Chitosan, CM-TMC, and Pam(3)Cys-functionalized CM-TMC polymers displayed cytotoxicity on mTHP1 cells in a concentration-dependent manner, which decreased by 50-fold on complexation with pDNA. In addition, decorated pDNA nanocarriers induced IL-8 secretion by mTHP-1 macrophages, which was increased by 10-fold as compared to nondecorated carriers.