Therapeutic ultrasound-mediated DNA to cell and nucleus: bioeffects revealed by confocal and atomic force microscopy

Abstract

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 (480%). 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.

Key factors that affect sonoporation efficiency in in vitro settings: The importance of standing wave in sonoporation

“…Although there are many reports on sonoporation [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38], each experimental setting differs from one another and the reported sonoporation efficiency, which is one of the most significant information in sonoporation, varies. In order to understand the effect of the experimental settings themselves on sonoporation experiments, we first investigated the impact of the conditions of the cultured cells on sonoporation efficiency.…”

“…Some reports, including our previous reports, show only a small amount of successfully sonoporated cells accompanying a large number of non-viable cells [25,26,30,31,35]. Others report otherwise [34,38]. Although some reports have pursued the possibility of optimizing ultrasound parameters [26,35,39,40], there has been no report on investigating how the technical differences of experimental settings can impact the results.…”

“…(Current address: Department of Neurosurgery, Osaka University Medical School, 2-2 Yamadaoka, Suita, Oaska, 565-0871, Japan. Phone: +81-6-6879-3652: FAX: +81-6-6879-3659; e-mail : m-kinoshita@nsurg.med.osaka-u.ac.jp) hyperthermia, such as coagulation of tissues [1][2][3][4][5][6][7][8][9][10][11][12], mechnical tissue destruction [13,14], disruption of tight junctions of the vasculature for enhanced local drug delivery [15][16][17][18][19][20], and sonoporation, a transient enhanced permeability of the cell membrane caused by the combination of ultrasound and microbubbles [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38]. As all of these bio-effects can be created non-invasively in vivo, ultrasound is gathering a great attention as a next generation surgical or drug delivery system.…”

Gene therapy progress and prospects: Ultrasound for gene transfer

“…Recently, however, Machluf's group have reported that exposing baby hamster kidney cells to 0.16 MI 1 MHz ultrasound for 30 min in the presence of Optison MCB yielded more rhodamine-labelled plasmid in the nucleus compared with a 10 min exposure. 22,23 Whether this truly reflects a direct effect of prolonged USE upon nuclear uptake is difficult to assess as there was also more plasmid in the cytoplasm after USE for 30 min, which clearly altered the plasmid concentration gradient from cytoplasm to nucleus compared with a 10 min USE. Of note was the observation that some Optison MCB remained intact after 30 min, again consistent with, but not proof of, a role for stable cavitation in promoting UEGT.…”

Use of ultrasound to enhance nonviral lung gene transfer in vivo

“…26 A recent study also demonstrated that the effects of US on the cell membrane are much more profound in the presence of Optison, providing further support for the role of cavitation. 27 Thus, we have tested, for the first time, the effect of sonoporation on nonviral gene transfer to the lung. It is well established, that ultrasound is greatly attenuated when travelling through air.…”