Applications of Quantum Dots in Gene Therapy

• Other Authors: Barnes, Laura F. (authoraut)
• Format: Others
• Language: English; English
• Published: Florida State University
• Subjects: Chemistry; Physical sciences; Mathematics
• Online Access: http://purl.flvc.org/fsu/fd/FSU_migr_etd-5467

Gene therapy is a rising field and requires multifunctional delivery platforms in order to overcome the cellular barriers. Quantum dots (QDs) provide a optically fluorescent and biocompatible surface to act as a multifunctional delivery platform for gene therapy. The objective of this research is to manipulate the surface of quantum dots for use in gene therapy. The first goal was to make the QDs water soluble and therefore biocompatible. The second goal was to functionalize the surface of the QDs with plasmid DNA for direct use in gene therapy. This approach uses chemoselective coupling chemistry between an InP/ZnS quantum dot (QD) and linker DNA (DNAlinker) to control the timing of protein expression. Linear DNA (lDNA), containing the CMV promoter and DsRed-Express gene, was condensed on the surface of the QD-DNAlinker. Optical and flow cytometry analysis of the DsRed-Express expression after transfection of the QD-lDNA into CHO cells shows a delayed protein expression for both coupling chemistries compared to naked lDNA. It is also clear that the protein expression form the QD-S-lDNA turns on quicker than the QD-NH-lDNA. We believe the protein expression delay is due to the site of coupling between the QD and DNAlinker and its affect on the lDNA packing strength. The S-DNAlinker is believed to couple by direct exchange at the vertices of the QD whereas the NH-DNAlinker couples through a condensation reaction to the facets. The delay in protein expression reflects the delayed exchange rate at the facets over the vertices. The ability to control the coupling chemistry and timing of release from the QD surface suggests a mechanism for dose control in transient gene therapeutics, and show QD delivery approaches are ideal candidates for multifunctional, targeted, drug carrying platforms that can simultaneously control dosing. The third goal of this research was to functionalize the surface of the QDs with the HIV cell penetrating peptide, TAT, and study its affects on QD internalization as well as toxicological affects within the cells. Tracking of the cellular uptake of these QDs by optical microscopy shows rapid, diffuse accumulation of both 10 % TAT and 100 % TAT passivated QDs throughout the cytosol of the cells. Toxicity studies were conducted by flow cytometry to investigate the effects of these materials on apoptosis, necrosis, and metabolic damage in Chinese Hamster Ovary (CHO) cells. These studies suggest toxic effects of the cell penetrating QDs are dependent on the amount of CAAKA-TAT used on the surface of the QD as well as the concentration of QD added. These observations aid in the use of QDs as self transfecting, nano delivery scaffolds for drug or gene therapy. === A Thesis submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Master of Science. === Summer Semester, 2010. === April 23, 2010. === Gene Therapy, Cell Penetrating Peptides, Bioconjugated Quantum Dots === Includes bibliographical references. === Geoffrey Strouse, Professor Directing Thesis; Timothy Logan, Committee Member; Brian Miller, Committee Member.