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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Therapeutic ultrasound promotes plasmid DNA uptake by clathrin-mediated endocytosis

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Author(s):
Bentes de Paula, Daisy Maria ; Valero-Lapchik, Valderez Bastos ; Paredes-Gamero, Edgar Julian ; Han, Sang Won [1]
Total Authors: 4
Affiliation:
[1] Univ Fed Sao Paulo, Lab CINTERGEN, Dept Biophys, BR-04044010 Sao Paulo - Brazil
Total Affiliations: 1
Document type: Journal article
Source: JOURNAL OF GENE MEDICINE; v. 13, n. 7-8, p. 392-401, JUL-AUG 2011.
Web of Science Citations: 28
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. Conclusions The present data indicate that plasmid DNA uptake promoted by US should occur via clathrin-mediated endocytosis. Copyright (C) 2011 John Wiley \& Sons, Ltd. (AU)