Date of Graduation

Summer 2020

Degree

Master of Science in Education in Secondary Education in Biology

Department

Graduate College

Committee Chair

Kyoungtae Kim

Abstract

Gene therapy is a very challenging field, especially with new emerging genetic disorders. Chitosan (CS), due to chitosan’s flexibility, biocompatibility, and biodegradability, has been of interest in the world of gene therapy especially as researchers are gravitating towards non-viral vectors due to the problems caused by viral vectors. Nevertheless, there are still issues regarding solubility, cellular uptake of cargos being transported in vitro or in vivo, increased cytotoxicity levels, as well as many other things that prevent chitosan from being an efficient gene delivery agent. Here I present five derivatives of chitosan, which were all modified with either triethylphosphonium butanamide (TEPB) or triethylammonium butanamide. In addition to the TEAB and TEPB groups, only two were modified with methoxy-poly (ethylene glycol) or mPEG units. The five derivatives are TEAB1-CS (24%of CS modified with TEAB), TEAB2-CS (40.6% TEAB), TEAB-mPEG-CS (40.6% TEAB; 3.1% mPEG), TEPB-mPEG-CS (43% TEPB; 2.6% mPEG), and TEPB-CS (43% TEPB). Cell proliferation and cytotoxicity assays were performed for the derivatives using an XTT assay kit which demonstrated mild to no defects on the growth of the cells. All derivatives were complexed with a TPST1-EGFP plasmid at a ratio of 10:1 and were able to transfect HeLa cervical cancer cells with varying degrees of efficiency. TEAB-mPEG-CS and TEPB-mPEG-CS had low cell viability at 100 mg/mL, however only TEPB-mPEG-CS induce apoptosis in a dose dependent manner. TEAB2-CS and TEPB-CS had low viability from the concentration ranges of 10-100 mg/mL and both induced apoptosis in a dose dependent manner. In particular, TEAB1-CS promoted viability at all treatment concentrations and showed the highest transfection efficiency among all 5 CS derivatives, while TEAB-mPEG-CS, TEPB-mPEG-CS, TEPB-CS, and TEAB2-CS exhibitited similar transfection efficiencies, suggesting that TEAB1-CS would be the most effective for gene transfection.

Keywords

chitosan nanoparticles, triethylammonium-butanamide chloride, triethylphosphonium-butanamide chloride, gene therapy, degree of quaternization, N+/P- ratio, P+/P- ratio, biopolymer cytotoxicity, pDNA transfection

Subject Categories

Biochemistry | Cancer Biology | Cell Biology | Medicinal-Pharmaceutical Chemistry | Molecular Biology | Other Chemistry

Copyright

© Deborah C. Ehie

Download

Open Access

Share

COinS