doi:10.1016/j.ultrasmedbio.2008.10.013 ● Original Contribution SONOPORATION BY ULTRASOUND-ACTIVATED MICROBUBBLE CONTRAST AGENTS: EFFECT OF ACOUSTIC EXPOSURE PARAMETERS ON CELL MEMBRANE PERMEABILITY AND CELL VIABILITY RAFFI KARSHAFIAN,* PETER D. BEVAN,* ROSS WILLIAMS, † SANYA SAMAC, † and PETER N. BURNS ,† *Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada; and † Imaging Research, Sunnybrook Health Sciences Centre, Toronto, ON, Canada (Received 2 June 2008; revised 13 October 2008; in final form 24 October 2008) Abtract—This work investigates the effect of ultrasound exposure parameters on the sonoporation of KHT-C cells in suspension by perflutren microbubbles. Variations in insonating acoustic pressure (0.05 to 3.5 MPa), pulse frequency (0.5 to 5.0 MHz), pulse repetition frequency (10 to 3000 Hz), pulse duration (4 to 32 s) and insonation time (0.1 to 900 s) were studied. The number of cells permeabilised to a fluorescent tracer molecule (70 kDa FITC-dextran) and the number of viable cells were measured using flow cytometry. The effect of exposure on the microbubble population was measured using a Coulter counter. Cell viability and membrane permeability were found to depend strongly on the acoustic exposure conditions. Cell permeability increased and viability decreased with increasing peak negative pressure, pulse repetition frequency, pulse duration and insonation time and with decreasing pulse centre frequency. The highest therapeutic ratio (defined as the ratio of permeabilised to nonviable cells) achieved was 8.8 with 32  4% permeabilisation and 96  1% viability at 570 kPa peak negative pressure, 8 s pulse duration, 3 kHz pulse repetition frequency, 500 kHz centre frequency and 12 s insonation time with microbubbles at 3.3% volume concentration. These settings correspond to an acoustic energy density (E SPPA ) of 3.12 J/cm 2 . Cell permeability and viability did not correlate with bubble disruption. The results indicate that ultrasound exposure parameters can be optimized for therapeutic sonoporation and that bubble disruption is a necessary but insufficient indicator of ultrasound-induced permeabilisation. (E-mail: raffik@sri.utoronto.ca) © 2009 World Federation for Ultrasound in Medicine & Biology. Key Words: Sonoporation, Cell membrane permeabilisation, Drug delivery, Microbubble, Ultrasound exposure. INTRODUCTION The impact of a medicinal drug depends on the rate and extent to which its constituents penetrate tissues and cell membranes to reach their intended target. Effectiveness is limited by side effects exerted by the drug on tissues and cells not associated with disease. Ideally, the drug should be delivered entirely to the diseased tissue, spar- ing healthy tissues. Existing modes of drug delivery, including oral, nasal, transdermal and i.v. administra- tions, are limited by various biological barriers obstruct- ing the pathway to the target, including the stomach, liver, kidney and, eventually, blood vessels and cell membranes (Orive et al. 2003). Cancer treatments in particular are constrained by inefficient intracellular de- livery of anticancer drugs (Jain 2001; Jain et al. 2007). A number of delivery methods, ranging from external to biochemical to invasive (Allen and Cullis 2004; Moses et al. 2003; Unger et al. 2004), have been developed with the aim of increasing local uptake of drugs; they have in common the requirement to transport agents to their site of action at sufficient concentrations in a safe and repro- ducible manner (Minchinton and Tannock 2006). Exposure to ultrasound energy has long been known to produce a variety of biological effects in tissues (Miller et al. 1996). Currently, ultrasound is used as a therapeutic modality in physiotherapy, lithotripsy and more recently in tumour ablation (Yu et al. 2004). It has also been shown that ultrasound, alone or in combination with externally administered microbubbles, can enhance transportation of macromolecules, including drugs and Address correspondence to: Raffi Karshafian, Department of Medical Biophysics, University of Toronto, 2075 Bayview Avenue, ST 39, Toronto, ON, Canada, M4N-3M5. E-mail: raffik@sri.utoronto.ca Ultrasound in Med. & Biol., Vol. 35, No. 5, pp. 847– 860, 2009 Copyright © 2009 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/09/$–see front matter 847