Quantitative Three-Dimensional Analysis of the Intracellular Trafficking of Plasmid DNA Transfected by a Nonviral Gene Delivery System Using Confocal Laser Scanning Microscopy

Abstract

Since endosomal escape and the nuclear delivery of plasmid DNA (pDNA) constitute major barriers for transgene expression, a quantitative evaluation of intracellular trafficking of pDNA would be highly desirable in terms of optimizing a nonviral gene delivery system. In the present study, a novel strategy is proposed for the quantification of rhodamine-labeled pDNA in endosomes/lysosomes, cytosol, and nucleus. Endosomes/lysosomes and nucleus were stained with LysoSensor DND-189 and Hoechst 33258, respectively, to distinguish them from the cytosol. The pixel areas of the clusters derived from the rhodamine were used as an index for the amount of pDNA. This approach was applied to the analysis of the intracellular trafficking of pDNA transfected by LipofectAMINE PLUS, stearylated octaarginine (STR-R8), and octaarginine (R8). In the case of R8, most of the pDNA was trapped by endosomes/lysosomes. STR-R8 exhibited endosomal escape followed by nuclear translocation in a time-dependent manner. LipofectAMINE PLUS was the most effective in rapidly delivering pDNA to the nucleus as well as the cytosol. These differences in the intracellular trafficking of pDNA correlated well with the transgene expression. Therefore, this method enables the quantitative analysis of the intracellular pharmacokinetics of pDNA and promises to provide useful information for optimizing nonviral gene delivery systems.

Non-linear pharmacodynamics in a non-viral gene delivery system: Positive non-linear relationship between dose and transfection efficiency

“…When nuclear fractionation was performed without treatment with CellScrub buffer, the percent of nuclear association of pDNA to the total cellular uptake at 1 h was estimated to be more than >70%, when transfected with LFN. This is inconsistent with our recent results showing that the average nuclear association was approximately 20%, when estimated by confocal image-assisted 3-dimensionally integrated quantification (CIDIQ) [2].…”

“…We recently, developed a quantitative method for evaluating each intracellular PK step, including internalization, endosomal escape, the nuclear delivery of a non-viral gene delivery system, using confocal laser scanning microscopy [2]. The findings showed that Lipofectamine PLUS (LFN) was capable of efficiently delivering plasmid DNA (pDNA) to cells via endocytosis, escape from endosomes, reaching the nucleus within 1 hr after transfection, which corresponds to the rate reported for Adenovirus [2].…”

The Applications of Metallic Nanowires for Live Cell Studies

“…Another trend observed in this experiment was that the uptake efficiency for the longer www.intechopen.com nanowire was lower than the shorter one except the 0.58 μm long gold nanowires, which were too short to determine the number of nanowires accurately. This trend agreed with the previous measurement using smaller nanorods (Bauer et al, 2003). Since nanowires with different aspect ratios exhibited the same cytotoxicity and the uptake efficiency of the nanowires decreased as the aspect ratio increased.…”

“…As for the surface modification of nanorods or nanowires, it is possible to selectively modify the multi-segment nanorods and nanowires. For example, selective surface modification scheme has been employed to modify two component nickel-gold nanowires to achieve bifunctionality (Bauer et al, 2003). In this case, the nanowire were reacted with 11-aminoundecanoic acid and 1,9-nonanedithol.…”

Octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery

“…However, complexes formed between plasmid DNA (pDNA) and R8 or STR-R8 peptides are internalized via classical endocytosis and become trapped in endosomes. 12,34 This suggests that the topology of R8 on a particle surface has a significant effect on the mechanism by which the particle enters the cell. Similar observations were made previously regarding the impact of the topology of the fusogenic peptide GALA on endosomal escape.…”