UC Irvine

Drug-resistant infectious bacteria are a pressing societal challenge with conventional antibiotics are becoming less effective at treating bacterial infections. More specifically, gram-negative intracellular infection provides an additional challenge. An innovative therapy for combating these emerging superbugs requires a paradigm-shift in therapy development. Therefore, a combinatory therapy of nanoantibiotics and gene therapy was developed. Chitosan, has been heralded as a material with applications in gene therapy, antimicrobials, wound healing, and various biomedical fields due to its availability, nontoxicity, and biodegradability, therefore, it was a highly suitable nanoantibiotic. However, the efficacy of chitosan is restricted due to the lack of aqueous solubility and limited response to biological triggers.

The focus of my work has been to engineer chitosan as an improved gene delivery vector while maintaining the antimicrobial properties. This was achieved by conjugating an amino branch to the primary hydroxyl group via a temporary, acid-cleavable ketal linkage to created acid-transforming chitosan (ATC). The antimicrobial capabilities of ATC were explored, and the possible mechanism of action was determined using transposon insertion sequencing. The efficacy of the free polymer was observed against S. typhimurium, an intracellular pathogen, alone at varying pH conditions which lead to a positive correlation between pH and effects the polymer had on the microbe. Additionally, ATC polyplexes were applied to RAW 264.7 cells infected with S. typhimurium expressing GFP and resulted in a decrease in the levels of bacteria found within the cells as confirmed by flow cytometry. This highlighted the key advantage of using an antimicrobial vector against difficult intracellular infections.

The temporary conjugated aminoethoxy branch onto chitosan increased the hydrophilicity, promoted cytosolic release in the mildly acidic endosome. The change in hydrophilicity and solubility of ATC was confirmed by dissolving ATC in deionized water and observing a complete dissipation. Acid-triggered reduction of ATC to native chitosan was achieved by incubation at an endosomal pH 5.0, and analyzed by NMR, MALDI-TOF, and FTIR. Improved cytosolic release, which led to improvement in gene delivery, was confirmed by the complexation of ATC with pDNA, and siRNA to form acid-responsive DNA/ATC (D/ATC) and siRNA/ATC (R/ATC) nanoparticles (NPs), The results indicated transfection of 30% and silencing of 85%, for both D/ATC and R/ATC, respectively.

In summary, this dissertation provides a comprehensive study from synthesis to application of acid-transforming chitosan as a potential synergistic treatment of drug-resistant microbes.