Therapeutic ultrasound-mediated DNA to cell and nucleus: bioeffects revealed by confocal and atomic force microscopy.
Therapeutic ultrasound (TUS) has the potential of becoming a powerful nonviral method for the delivery of genes into cells and tissues. Understanding the mechanism by which TUS delivers genes, its bioeffects on cells and the kinetic of gene entrances to the nucleus can improve transfection efficiency and allow better control of this modality when bringing it to clinical settings. In the present study, direct evidence for the role and possible mechanism of TUS (with or without Optison) in the in vitro gene-delivery process are presented. Appling a 1 MHz TUS, at 2 W/cm(2), 30%DC for 30 min was found to achieve the highest transfection level and efficiency while maintaining high cell viability (>80%). Adding Optison further increase transfection level and efficiency by 1.5 to three-fold. Confocal microscopy studies indicate that long-term TUS application localizes the DNA in cell and nucleus regardless of Optison addition. Thus, TUS significantly affects transfection efficiency and protein kinetic expression. Using innovative direct microscopy approaches: atomic force microscopy, we demonstrate that TUS exerts bioeffects, which differ from the ones obtained when Optison is used together with TUS. Our data suggest that TUS alone affect the cell membrane in a different mechanism than when Optison is used.
Duvshani-Eshet
M
M
The Faculty of Biotechnology and Food Engineering, The Technion - Israel institute of Technology, Haifa.
Baruch
L
L
Kesselman
E
E
Shimoni
E
E
Machluf
M
M
D000418:Albumins / Q000627:therapeutic use; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D002477:Cells / Q000378:metabolism*; D003287:Contrast Media; D004247:DNA / Q000008:administration & dosage* / Q000032:analysis; D005466:Fluorocarbons / Q000627:therapeutic use; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D018625:Microscopy, Atomic Force; D018613:Microscopy, Confocal; D008863:Microspheres; D014162:Transfection / Q000379:methods*; D014464:Ultrasonic Therapy
D000418:Albumins; D003287:Contrast Media; C099458:FS 069; D005466:Fluorocarbons; D049452:Green Fluorescent Proteins; D004247:DNA