Chitosan has been shown to have antimicrobial and gene delivery capabilities. However, lack of solubility in various solvents and media as well as poor responsiveness to biological triggers have limited its use in biomedical applications, which has been tried to overcome by various methods such as N-O-carboxyalkyl modification or acylation. In this study, we attempted to increase the chitosan's aqueous solubility and stimuli-responsiveness by conjugating an amino branch to the primary hydroxyl group of chitosan via an acid-cleavable ketal linkage. It was hypothesized that the addition of amine group onto chitosan would increase its hydrophilicity while the acid-cleavable ketal linkage would promote the cytosolic release of the payload upon the hydrolysis in the mildly acidic endosome. The acid-hydrolysis of the amino ketal branch generates the chitosan in its native, antimicrobial form. To synthesize the acid-responsive chitosan, the primary hydroxyl group on chitosan was conjugated with 2-(2-aminoethoxy) ethanol by forming dimethyl ketal linkage. The synthesized ketalized chitosan (kC) was characterized for ketalization ratio and molecular weight by NMR, FTIR, and GPC. Acid-triggered reduction of kC to native chitosan was also confirmed after an incubation at an endosomal pH 5.0. GFP plasmid DNA and siRNA were complexed with kC at varying N/P ratios to form acid-responsive DNA/kChitosan (D/kC) and siRNA/kChitosan (R/kC) nanoparticles (NPs). The resulting D/kC NPs and R/kC NPs were characterized for size and surface charge by dynamic light scattering (DLS) and zeta-potential measurement, respectively. Transfection efficiency and cytotoxicity of D/kC NPs and R/kC NPs were assessed by incubating them with HeLa and HeLa/GFP cells, respectively, followed by measuring GFP expression levels using flow cytometry. The results indicated significantly improved transfection in comparison to unmodified chitosan, for both D/kC and R/kC. Next subsequent studies include the investigation of antimicrobial effects of the nucleic acid/kC NPs on various drug resistant microbes. This talk will present the rationale, the design, and the feasibility of a novel gene delivery vector for synergistic treatment of drug-resistant microbes.

Acid-responsive chitosan as efficient gene delivery carriers and nanoantibiotics materials

248th National Meeting (2014)

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