Combination of differential interference contrast with prism-type total internal fluorescence microscope for direct observation of polyamidoamine dendrimer nanoparticle as a gene delivery in living human cells

摘要

A combined system of differential interference contrast (DIC) and total internal reflection fluorescence microscope (TIRFM) with a transmitted all-side polished dove prism was used for the direct monitoring of polyamidoamine (PAMAM) dendrimer nanoparticles as a gene delivery in living human embryonic kidney 293 (HEK 293) cells. The PAMAM dendrimer conjugated with fluorescein isothiocyanate (FITC) was used to form a fluorescent nanoparticle with the plasmid DNA (complexes) in order to directly monitor the entry of the complexes inside living cells. The DIC image provided precise information of the living HEK 293 cellular structures. Without moving the cell, the TIRFM images of the PAMAM nanoparticle-DNA complexes on the all-side polished dove prism provided precise information on the distance between the cell membrane and the complexes (< 200 nm) as well as the real-time localization of the individual complexes in the cells. The complexes were observed in cytosol within 4 h after incubating the cells with the complexes in Dulbecco's modified eagle's medium. The localization data of the complexes inside the cell obtained by TIRFM were reconfirmed using 3D confocal microscopy images of the complexes at the subcellular localization. These results suggest that the combined system of DIC and all-side polished dove prism-type TIRFM is a powerful tool for the direct real-time monitoring of the internalization and subcellular localization of nanoparticles carrying genes through a nonviral approach for gene therapy.