Title

Quantum dot assisted long-term intracellular trafficking and development of safe and efficient non-viral vector

Date of Completion

January 2008

Keywords

Chemistry, Pharmaceutical

Degree

Ph.D.

Abstract

A number of non-viral DNA delivery systems and transfection protocols are under investigation. However, these show limited transfection efficiency and some are toxic to cells. The research described herein was aimed at developing novel methods to investigate potential roadblocks to the trafficking of non-viral DNA delivery systems and to develop novel and safer non-viral delivery systems. ^ As a novel approach to assessing intracellular DNA stability, plasmid DNA was dual labeled with rhodamine and fluorescein at two different sites. Gel-electrophoresis and time-lapse confocal microscopy confirmed visualization of DNA as a yellow color due to the close proximity of two fluorophores and separation of colors in the event of strand break. Thus color separation is a probable indication of DNA degradation inside cells. ^ To overcome photo-instability limitations of organic fluorophores, quantum dots (QDs) conjugated plasmid DNA was utilized, for long-term single cell tracking studies. Plasmid DNA-QD labeling via peptide nucleic acid linkers was facilitated by encapsulating cadmium selenide/zinc sulphide QDs in maleimide functionalized PEG lipids. QD-DNA labeling was confirmed by atomic force microscopy and gel electrophoresis. Transfection efficiencies of QD-DNA conjugates were comparable to unconjugated plasmid DNA demonstrating no loss in DNA functionality. Cellular uptake, cytoplasmic and nuclear distribution of QD-DNA conjugates were evaluated using confocal microscopy. QD-DNA conjugates were non-toxic as revealed by cytotoxicity assay and provide a unique tool for investigation of intracellular trafficking. ^ As an approach to develop a safe and efficient DNA delivery system, anionic liposomal systems formed by complexing 1,2-dioleoyl-sn-glycero-3-phospho-glycerol/1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (anionic/zwitterionic lipid) liposomes with plasmid DNA and Ca2+ were developed and optimized for high transfection efficiency and low cytotoxicity. Particle size analysis, gel electrophoresis, transmission electron microscopy and confocal studies assisted in characterization of optimized formulations. Maximum transfection efficiency occurred at 15-20 mM Ca2+ and 20/80 (anionic/zwitterionic lipid) and at high lipid/DNA ratios, 15–20 μg/0.8 μg. The transfection efficiency and cell viability of anionic lipoplexes in serum were ∼78% and ∼93%, respectively, which was comparable to cationic lipoplexes, which had transfection efficiency and cell viability values of ∼68% and ∼35%, respectively. Thus anionic lipoplexes appear to be suitable candidates for DNA delivery. ^