Biochemical Characterization of Polycationic Nucleic Acid Delivery Vectors
Herein, four classes of polycationic nucleic acid vectors were investigated for their interaction in the complex biological environment of live cells: Poly(glycoamidoamine)s (PGAA)s, lanthanide-containing polycations (La-polycations), PEG-star peptides, and b-cyclodextrin click clusters (b-CD clusters). Core work in this dissertation concentrates on the intracellular trafficking mechanisms of polycationic vectors. The long-term goal of this project is to aid the future design of vectors that interact with biological molecules in a precise, predictable, and beneficial way.
The (PGAA)s have decreased polycationic density in comparison to traditional polycationic vectors resulting in prolonged vesicular transport and efficacious intracellular delivery. To understand why their structure produces these results, trafficking mechanisms are investigated using intracellular pH assays, live-cell time-lapse confocal microscopy, and ICC/IF experiments. Furthermore, La-polycations inherently permit imaging of the vector via fluorescence or magnetic resonance imaging, which is extremely relevant to future in vivo and clinical trials. Studies in this dissertation reveal information about the co-localization between La-polycation and pDNA as well as shed light on mechanisms of nuclear import. The final two classes of vectors (PEG-star peptides and b-CD clusters) are multivalent vectors. Research herein demonstrates that both multivalent architectures substantiate a bridging transport mechanism. That is, one end of the vector can bind DNA at the core of the polyplex, while simultaneously binding cell surface molecules.
In conclusion, the importance of investigating the biological impact of these structures is crucial to advancing the field of nonviral nucleic acid delivery. Of most impact, research in this dissertation demonstrates the proton sponge mechanism, which is the only widely accepted mechanism for endocytic escape, is not globally applicable. My work supports another theory of vesicular escape based on the extended delivery to other organelles and degradation properties of polycations. Furthermore, the La-polycations reveal the power of lanthanide-based imaging to elucidate transfection pathways. Finally, multivalent vectors evidence a bridging transport mechanism which may increase cellular uptake without the need for polycations of increased charge density. Overall, the combination of chemical moieties such as those studied herein may lead to new generation of effective supramolecular vectors.
School:University of Cincinnati
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:nonviral gene delivery confocal microscopy lanthanide imaging
Date of Publication:01/01/2008