Therapeutic ultrasound promotes plasmid DNA uptake by clathrin‐mediated endocytosis

Abstract

Background Ultrasound (US) has been widely used to improve the efficiency of nonviral vector transfection. The mechanism of plasmid uptake is usually attributed to sonoporation, although there is not clear evidence for this attribution. Based on our previous results, we hypothesized that other mechanisms, such as endocytosis, could be involved in this process.Methods NIH3T3 cells were transfected with plasmid vector pEGFP-N3 (4.7kb) using a therapeutic US without microbubbles. Bioeffects such as calcium influx, reactive oxygen species (ROS) generation and membrane potential alterations were accessed with fluorescent dyes in real-time by confocal microscopy after US insonation. Localization of labeled plasmid DNA in cells was also monitored with endocytosis markers using an immunofluorescence assay.Results US at 2 W/cm 2 with a duty-cycle of 20% for 30 s resulted in approximately 40% transfection efficiency but, at 1 W/cm 2 , resulted in a very low level of transfection. Both the production of ROS and calcium influx were augmented during the insonation, although they were stopped soon after turning off US, with the exception of calcium influx with 1 W/cm 2 . US also changed the cell membrane potential to the hyperpolarization state, which returned to the normal state soon after insonation. Labeled plasmids DNA could be co-localized with clathrin-mediated endocytosis marker but not with caveolin-1. ConclusionsThe present data indicate that plasmid DNA uptake promoted by US should occur via clathrin-mediated endocytosis. Figure 2. Representative images of NIH3T3 cells transfected by US. Cells were transfected with 20mg of pEGFP-N3 and US parameters were adjusted to 20% DC and 2 W/cm 2 for 30 s. Efficiency of transfection in other conditions can be seen in the Tables 1, 2 and 3. Fluorescent micrograph of cells expressing green fluorescent protein 48 h after of transfection (a). Micrograph without fluorescence of the cells in (b).Plasmid DNA endocytosis induced by ultrasound 395 using 20% DC for 30 s at 1 W/cm 2 (a) and 2 W/cm 2 (b). Graphs show calcium influx at different time points. The two dotted lines represent when the US was switched on. F t /F 0 is the ratio of total fluorescence to fluorescence at time zero. Plasmid DNA endocytosis induced by ultrasound 397 DAPI dye. The images represent the responses of at least three independent experiments.Plasmid DNA endocytosis induced by ultrasound 399

Understanding ultrasound induced sonoporation: Definitions and underlying mechanisms

“…As mentioned above, the low frequency ultrasound in the kHz range used in these studies promotes the generation of gas nuclei via rectified diffusion, which can subsequently implode and provoke the same bioeffects as imploding microbubbles. In contrast to these publications, other authors have suggested endocytosis as mechanism when using ultrasound in the absence of microbubbles [101][102][103].…”

Ultrasound and microbubble mediated drug delivery: Acoustic pressure as determinant for uptake via membrane pores or endocytosis

“…Molecule size was observed to determine decisively which route of uptake occurs, with endocytosis playing a greater role for larger molecules. A few other studies confirmed that endocytosis is indeed involved in ultrasound mediated uptake [15][16][17][18]. In contrast, Schlicher et al [19] blocked endocytic routes and found no difference in uptake of calcein.…”

Hydrogen peroxide enhances the uptake of polyethylenimine/oligonucleotides complexes in A549 cells by activating CaMKII independent of [Ca2+]c elevation

“…Paula et al (2011) reported that the enhanced efficiency of non-viral vector transfection was relative to ROS production using a therapeutic ultrasound. However, the role of ROS in the uptake of PEI/gene particles in cells remains unknown.…”

“…ROS are increased in a variety of lung diseases (Wood et al, 2010). Enhanced transfection has been related to the extent of ROS production after stress stimuli (Paula et al, 2011); however, the role of ROS in the uptake of PEI/gene particles in cells remains unknown.…”