Conventional antibiotics are becoming less effective at treating certain bacterial infections. Most have resulted in bacterial resistances through efflux pumps, modifying/inactivating enzymes, or mutations at the drug target site. Therefore, effective therapy for combating these emerging superbugs requires innovative, techniques that combines conventional antimicrobial therapies with silencing drug-resistance processes in microbes. Chitosan has been heralded as a material with applications in gene therapy, antimicrobials, wound healing, and various biomedical fields. However, the widespread use of chitosan is restricted due to the lack of solubility in neutral solutions and limited response to biological triggers. We attempted to increase chitosan’s aqueous solubility and stimuli-responsiveness by conjugating an amino branch to the primary hydroxyl group via a temporary, acid-cleavable ketal linkage. We hypothesized that the advantage of the temporary amine group onto chitosan would 1) increase the hydrophilicity, 2) promote cytosolic release in the mildly acidic endosome, and 3) regenerate chitosan in its native antimicrobial form upon acid hydrolysis. Acid-triggered reduction of (kC) to native chitosan was achieved by incubation at an endosomal pH 5.0, and analyzed by NMR, MALDI-TOF, and FTIR. To confirm the morphology of kC NPs, 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 NPs were characterized for size and surface charge by dynamic light scattering (DLS) and zeta-potential measurement, respectively. Transfection efficiency and cytotoxicity of the NPs were assessed by incubating them with HeLa cells and measuring GFP expression levels using flow cytometry. The results indicated significantly improved transfection in comparison to unmodified chitosan. Additionally, acid hydrolyzed kC NPs underwent morphological changes that resembled those of unmodified chitosan NPs, indicating pH responsive regeneration of native chitosan. We concluded that the change from acidic pH to biological pH in the cytosol, enhances the nuclei acid delivery capability of kC in comparison to native chitosan. This talk will discuss the engineering and efficacy of a polysaccharide based gene delivery vector for potential synergistic treatment of drug-resistant microbes.

Engineering pH-responsive chitosan nanoparticles as a genetically specific nanoantibiotic

251st National Meeting (2016)

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