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IC‐P‐005: In‐Vitro and In‐Vivo Study of a Magnetic Resonance Imaging (MRI)/Fluorescent Contrast Agent for Detection in Alzheimer's Disease
Author(s) -
Ta Robert,
Li Alex X.,
Suchy Mojmir,
Hudson Robert H.E.,
Pasternak Stephen H.,
Bartha Robert
Publication year - 2010
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2010.05.020
Subject(s) - magnetic resonance imaging , in vivo , chemistry , mri contrast agent , gadolinium , nuclear magnetic resonance , dota , fluorescence , nuclear medicine , medicine , radiology , biology , microbiology and biotechnology , organic chemistry , physics , quantum mechanics
uptake of this MRI/fluorescent contrast agent targeting CatD. Methods: SN56 cells, a neuronal cell line, were cultured and differentiated. To simulate conditions of increased CatD, cells were transfected with a plasmid which stimulates high level expression of human CatD tagged with red fluorescent protein. Varying concentrations (5, 10, 50, 100 mM) of DOTA-CAT were added to the media of SN56 cells and confocal microscopy was performed at 30 minutes. Uptake was quantified by counting the proportion of cells which were labeled with DOTA-CAT expressed as Mean 6 SEM. Lastly, cleavage experiments on DOTA-CAT were performed using purified CatD and analyzed using electrospray-ionization mass spectrometry (ESI-MS). Results: In uptake experiments, 19 6 2% of cells exposed to the agent at 5 mM took up DOTA-CAT, and this number increased with concentration such that at 100 mM, 78 6 7% of cells took up DOTA-CAT. In contrast, a maximum of 5 6 1% of cells not over-expressing CatD took up DOTA-CAT over the entire concentration range. DOTA-CAT was observed intracellularly and in intracellular vesicles frequently co-localized with red fluorescent protein tagged-CatD within lysosomes. In cleavage experiments, incubation of DOTA-CAT with CatD produced a 1906.6 Da fragment confirming the cleavage of DOTA-CAT at the predicted CatD recognition sequence. Conclusions: These experiments demonstrate that DOTA-CAT interacts with CatD and is cleaved as predicted. Furthermore, DOTA-CAT is preferentially taken up by SN56 cells over-expressing CatD in a concentration dependant manner. Therefore, DOTA-CAT shows significant potential as a molecular imaging probe that will be further evaluated in future experiments using an in-vivo model of AD.