Physical parameters influencing optimization of ultrasound-mediated DNA transfection.
Ultrasound (US) has been shown to transiently disrupt cell membranes and, thereby, facilitate the loading of drugs and genes into viable cells. To address optimization of gene therapy applications, the aim of this work was to systematically determine the influence of physical parameters on transfection and viability of DU145 prostate cancer cells by two different DNA plasmids (pEGFP-N1 and pGL3). By sonicating cells in vitro in the presence of naked DNA, we found that transfection efficiency was increased by: 1. optimizing acoustic energy at 10 to 30 J/cm(2) (for our apparatus, at pressures above the cavitation threshold); 2. using 500-kHz US in the presence of Optison to nucleate cavition, rather than 24-kHz US without Optison; 3. increasing cell concentration from 10(6) to 10(7) cells/mL; and 4. changing temperature during sonication from 21 to 37 degrees C. The best conditions in this study increased transfection by almost 100-fold in the absence of significant DNA damage. Additional measurements indicated that less than one fourth of cells with DNA plasmid uptake into the cytosol showed DNA expression, which suggests that further optimizing transfection by US may require facilitating intracellular DNA trafficking.
Zarnitsyn
Vladimir G
VG
School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, USA.
Prausnitz
Mark R
MR
D000418:Albumins / Q000494:pharmacology; D002452:Cell Count; D002470:Cell Survival / Q000235:genetics; D003287:Contrast Media / Q000494:pharmacology; D004247:DNA / Q000187:drug effects / Q000235:genetics*; D004249:DNA Damage / Q000235:genetics; D005466:Fluorocarbons / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D010957:Plasmids; D013010:Sonication; D013696:Temperature; D014162:Transfection; D014407:Tumor Cells, Cultured
D000418:Albumins; D003287:Contrast Media; C099458:FS 069; D005466:Fluorocarbons; D004247:DNA