Development and application of Tat-near-infrared-emissive polymersomes for in vivo optical imaging of dendritic cells
Advances in the understanding of disease at the cellular level and the ability to manipulate cells in culture have led to the development of cell-based therapeutics. In contrast to prior therapeutic regimes including small molecules, peptides, and full proteins, cells are highly complex and variable. The institution of cellular therapeutics requires optimization of many parameters and a thorough understanding of how each parameter alters the efficacy of the final therapeutic. This cumbersome task calls for new tools to enable high throughput investigations. The field of molecular imaging is positioned to provide the needed tools to enable cellular tracking technology. In particular, optical imaging is an emerging modality with many advantages for high throughput small animal studies. ^ In this thesis, recently developed near-infrared (NIR) emissive polymersomes are engineered for optimal cellular uptake to enable fluorescence-based cellular tracking. NIR-emissive polymersomes are polymeric vesicles formed from amphiphilic block co-polymers and near-infrared emissive porphyrin fluorophores. Directing cellular uptake of NIR-polymersomes is achieved by functionalizing the vesicle surface with a cationic peptide, namely Tat. Tat-NIR-polymersomes are subsequently used to label dendritic cells and evaluate in vivo tracking capabilities. ^ Dendritic cells are successfully labeled with Tat-NIR-polymersomes and characterized according to the degree of uptake, cell phenotype, viability and the label stability. The Tat peptide is shown to enhance cellular loading over non-functionalized vesicles. Neither reagent caused changes to cell phenotype or the ability to differentiate from immature to mature dendritic cells. ^ Two small animal fluorescence imaging instruments were employed in this work: GE/ART eXplore Optix and Licor Odyssey. In vitro and tissue phantom imaging studies established that the GE/ART instrument as the superior instrument for in vivo studies. In particular, this instrument enables both fluorescence intensity and fluorescence lifetime imaging. A battery of in vivo tracking studies were explored in this thesis with a focus on injection route, dendritic cell biodistributions and DC migration kinetics in lymph nodes. In addition, fluorescence lifetime imaging is proven to be an invaluable tool for differentiating NIR-DC fluorescence from background fluorescence. This dissertation establishes Tat-NIR-polymersomes as a viable material for cell tracking and, ultimately, cell therapy optimization. ^
Biology, Cell|Engineering, Biomedical|Health Sciences, Immunology
Natalie Aronson Christian,
"Development and application of Tat-near-infrared-emissive polymersomes for in vivo optical imaging of dendritic cells"
(January 1, 2008).
Dissertations available from ProQuest.