(205e) pH-Responsive Chitosan Nanoparticles As a Gene Therapy-Based Nanoantibiotic

Due to its availability, nontoxicity, and biodegradability, chitosan has been heralded as a material with applications in antimicrobials, manufacturing, and certain biomedical fields. However, certain factors limiting the widespread use of chitosan include the lack of solubility in neutral solutions and limited response to biological triggers. Attempts to overcome these shortcomings have led to various modifications such as N-/O-carboxyalkyl modification or acylation. In this study, we attempted to increase chitosan’s aqueous solubility and simultaneoulsy add stimuli-responsiveness by conjugating an amino branch to the primary hydroxyl group via a temporary, acid-cleavable ketal linkage, resulting in ketalized chitosan (kC). We hypothesized that enginered chitosan with amine branches via ketal linkage would 1) increase the hydrophilicity, 2) promote cytosolic release in the mildly acidic endosome, and 3) regenerate chitosan back in its native antimicrobial form upon acid hydrolysis. The increased hydrophilicity and solubility of kC was confirmed by aquesous dissolution of kC with a complete dissipation. Acid-triggered reduction of kC to native chitosan was confirmed by incubation it at an endosomal pH 5.0, and analyzed by NMR and FTIR. Further, kC was used to complex nucleic acids, GFP-encoding plasmid DNA or anti-GFP siRNA, for its potential use as a stimuli-responsive gene carrier. The size, surface charge, and morphology of nucleic acid/kC NPs prepared at varying N/P ratios, DNA/kChitosan (D/kC) and siRNA/kChitosan (R/kC) NPs, were measured and observed by dynamic light scattering (DLS), zeta-potential measurement, and transmission electron microscopy (TEM), respectively. Transfection and gene silencing, and cytotoxicity of D/kC 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 and gene silencing with minimal cytotoxicity, in comparison to unmodified chitosan, for both D/kC and R/kC. Additionally, acid-hydrolyzed kC NPs converted to the morphology resembling that of unmodified chitosan NPs, indicating pH-responsive regeneration of native chitosan. We also concluded that the change from acidic pH to physiological pH in the cytosol triggered the release of nucleic acids from kC.  To demonstrate the feasibility of using the nucleic acid/kC NPs in treating infectious diseases using a gene therapy approach, antimicrobial effects of the nucleic acid/kC NPs on Chlamydia trachomatis-infected cells is under investigation. This talk will discuss the engineering approach to develop an efficient and safe polysaccharide-based gene delivery vector for synergistic treatment of drug-resistant microbes.