Modulation control over ultrasound-mediated gene delivery: Evaluating the importance of standing waves Mariame A. Hassan a,b , Mikhail A. Buldakov c,d,e , Ryohei Ogawa a , Qing-Li Zhao a , Yukihiro Furusawa a , Nobuki Kudo f , Takashi Kondo a, ⁎, Peter Riesz g a Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical, Sciences, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan b Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Kasr Al-Aini str., Cairo 11562, Egypt c Cancer Research Institute of Tomsk Scientific Center, Siberian Department of Russian Academy of Medical Sciences, Kooperativnii str., b. 5, Tomsk 634009, Russia d Tomsk State University, Department of Physiology, pr. Lenina, b. 36, Tomsk 634050, Russia e Institute of High Current Electronic, Siberian Department of Russian Academy of Sciences, pr. Akademichaskii, b. 2/3, Tomsk 634055, Russia f Laboratory of Biomedical Instrumentation and Measurements, Graduate School of Information Science and Technology, Hokkaido University, Sapporo 060-0814, Japan g Radiation Biology Branch, National Cancer Institute, NIH, Bethesda, MD 20892-1002, USA abstract article info Article history: Received 15 June 2009 Accepted 23 August 2009 Available online 29 August 2009 Keywords: Gene delivery Low-intensity ultrasound Pulse repetition frequency (PRF) Ultrasound streaming Standing waves Low modulation frequencies from 0.5 to 100 Hz were shown to alter the characteristics of the ultrasound field producing solution agitation (b 5 Hz; region of “ultrasound streaming” prevalence) or stagnancy (N 5 Hz; region of standing waves establishment) (Buldakov et al., Ultrason. Sonochem., 2009). In this study, the same conditions were used to depict the changes in exogenous DNA delivery in these regions. The luciferase expression data revealed that lower modulations were more capable of enhancing delivery at the expense of viability. On the contrary, the viability was conserved at higher modulations whereas delivery was found to be null. Cavitational activity and acoustic streaming were the effecters beyond the observed pattern and delivery enhancement was shown to be mediated mainly through sonopermeation. To promote transfection, the addition of calcium ions or an echo contrast agent (Levovist ® ) was proposed. Depending on the mechanism involved in each approach, differential enhancement was observed in both regions and at the interim zone (5 Hz). In both cases, enhancement in standing waves field was significant reaching 16.0 and 3.3 folds increase, respectively. Therefore, it is concluded that although the establishment of standing waves is not the only prerequisite for high transfection rates, yet, it is a key element in optimization when other factors such as proximity and cavitation are considered. © 2009 Elsevier B.V. All rights reserved. 1. Introduction The issue of “gene therapy” feasibility is no longer the major issue that matters after a long list of DNA-based therapeutics have been introduced for clinical development as candidates for treating several malignant as well as non-malignant diseases [1]. Instead, searching for methods for optimizing “gene delivery” is now the most challenging branch of research. In that sense, ultrasound (US) has been proposed as a promising delivery aid in what is called “sonotransfection”. US is an inexpensive and convenient method that can localize drugs and macromolecules in cells not only in superficial tissues but also in deeper ones in a non-invasive manner. It has been ascertained that US facilitates the traverse of high molecular weight substances through the cell membrane through a process called “sonoporation” [2–5]. Also a role for US in aiding nuclear uptake has been proposed [6,7]. Recently, US has been shown to be useful in targeted drug delivery [8,9]. Despite these advantages, sonotransfection efficiency is still far below the desired levels and is accompanied by various degrees of deleterious effects on cells. There is extensive work being done in order to alleviate these drawbacks [10–12]. In fact, there are a lot of variables that contribute to the cellular response to US irradiation. These variables can be broadly divided into acoustic parameters which include the intensity, the frequency, and the mode of irradiation (pulsed or continuous), and non- acoustic variables which include the cell density, cell fashion during sonication (suspended/attached/spheroids), and the arrangement of the sonication setup which itself introduces more variables such as the distance and position of the transducer from the cells, the impedance of the transmitting medium and the type of exposure vessel. Under such conditions of multiple sources of variability, though general “optimiza- tion” may be very difficult, a better understanding of the role of each variable over a wide range is a prerequisite for achieving maximum clinical benefit. In a previous study, we have shown the dependence of the biological and chemical effects of US on pulse repetition frequency (PRF) at low intensities with considerable justification of these effects [13]. Briefly, we have concluded that the PRF range under study (from 0.5 to 100 Hz) Journal of Controlled Release 141 (2010) 70–76 ⁎ Corresponding author. Tel.: +81 76 434 7265; fax: +81 76 434 5190. E-mail address: kondot@med.u-toyama.ac.jp (T. Kondo). 0168-3659/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2009.08.020 Contents lists available at ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel GENE DELIVERY