Abstract C66: A novel technique to quantify condensation/decondensation status in the nuclear subdomains by QD-FRET

Abstract

Abstract Recent studies have reported that control of nuclear decondensation at specific nuclear subdomains (i.e. euchromatin) is highly desired for the efficient transfection efficiency. Here, we have established a novel technology for the imaging of nuclear condensation/decondensation status in the nuclear subdomains (i.e. heterochromatin and euchromatin) by based on the three key technologies. One is imaging of fluorescence resonance energy transfer (FRET) between quantum dots (QDs)-labeled plasmid DNA (pDNA) as a donor, and acceptor-labeled polycation, which can monitor a specific molecular interactions at the nanometre scale. The second technology is a multilayered lipid envelope-type nanoparticle, which we refer to as a Tetra-lamellar Multi-functional Envelope-type Nano Device (T-MEND). The critical structural elements of the T-MEND are a DNA—polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two endosome-fusogenic outer envelopes, which are designed for nuclear delivery via a stepwise fusion. It is required to evaluate the nuclear dispositions in relation to the transgene expression, to avoid a situation that intracellular trafficking processes rate-limit transfection activity. The last one is confocal image-assisted 3-dimensionally integrated quantification (CIDIQ) method. In this method, fluorophore-labeled pDNA was transfected in HeLa cell line, and then intracellular distribution was distinguished by staining organelle. Z-series of confocal images were captured by confocal laser scanning microscopy (CLSM), and thereafter, pDNA was quantified based on the pixel area of the signals derived from the fluorophore on pDNA in nuclear subdomains (heterochromatin and euchromatin). As a model of the analysis, QD-labeled DNA was condensed with reductive environment-dependently cleavable polyrotaxane (DMAE-ss-PRX), an artificial condenser, and encapsulated in T-MEND. As a result, transfection activity depends on the number of cationic DMAE of cyclodextrin (α-CD), in conjunction with a drastic change in decondensation efficiency in euchromatin region. These data collectively indicate that this imaging technology is highly advantageous to clarify the dynamics of plasmid DNA in post-nuclear delivery process of cancer cells. Citation Information: Cancer Res 2009;69(23 Suppl):C66.