Original contribution
Sonoporation by Ultrasound-Activated Microbubble Contrast Agents: Effect of Acoustic Exposure Parameters on Cell Membrane Permeability and Cell Viability

https://doi.org/10.1016/j.ultrasmedbio.2008.10.013Get rights and content

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 μs) 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 μs 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 (ESPPA) of 3.12 J/cm2. 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: [email protected])

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, sparing healthy tissues. Existing modes of drug delivery, including oral, nasal, transdermal and i.v. administrations, are limited by various biological barriers obstructing 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 delivery 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 reproducible 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 genetic materials, across cell membranes and blood vessels (Iwanaga et al 2007, Kinoshita and Hynynen 2005, Saito et al 2007, Wei et al 2004). One of the earliest investigations on the use of ultrasound to enhance drug delivery was reported by Fellinger and Schmid in 1954, when they enhanced the delivery of hydrocortisone ointment into inflamed tissue (Ng and Liu 2002). In 1995, Tachibana et al. reported that ultrasound in conjunction with microbubbles accelerated thrombolysis by urokinase. The phenomenon by which transient, nonlethal porosity in biological membranes is induced through ultrasound exposure is known as sonoporation or sonophoresis. The physical mechanisms underpinning sonoporation are not fully understood, however, effects associated with bubble disruption, such as the generation of shockwaves and microjets (Ohl et al. 2006) and with stable bubble oscillation, such as acoustic microstreaming (Marmottant and Hilgenfeldt 2003, van Wamel et al 2006, Wu 2007), are likely to be involved.

The advantage of ultrasound as a therapeutic system is that it can be focused with millimeter precision within the body to produce specific and localized effects. Ultrasound can be applied externally, intravascularly or endoscopically and can be used to locally release therapeutic agents from such carriers as liposomes and microbubbles (Unger et al. 2004). Microbubble agents can be loaded with therapeutic molecules and can be conjugated with ligands that bind to cell receptors, both for molecular imaging and for enhancing localized drug uptake (Dayton and Ferrara 2002, Lindner and Kaul 2001, Unger et al 2001, Unger et al 2004). Furthermore, the application of therapeutic ultrasound can be guided by a variety of imaging modalities, including ultrasound itself as well as magnetic resonance imaging (Hynynen and Clement 2007, Hynynen et al 2001).

An improved understanding of the interaction of ultrasound and microbubbles with tissue is important for the realization of clinically useful therapeutic methods and devices (Yu et al. 2004). To understand sonoporation, a required first step is to determine the impact of ultrasound exposure parameters and microbubble characteristics on the phenomenon. The published literature reports significant variations in the efficacy of ultrasound-induced permeabilisation of biological membranes (Guzmán et al 2001, Miller et al 1999, Miller et al 2002); however, the methods of ultrasound exposure and the experimental conditions vary greatly between investigators, making it difficult to compare results (Kinoshita and Hynynen 2007, Miller 2007).

The quantitative dependence of sonoporation on ultrasound exposure parameters is not well documented and is the subject of this study. We hypothesize that cellular uptake of a high molecular weight marker mediated by ultrasound exposure in the presence of microbubbles can be optimized to achieve high delivery efficiency and minimal cell death. The objectives of this study are to investigate systematically the effect of ultrasound exposure parameters on cell membrane permeability and viability and to identify optimum ultrasound exposure regimes for cell permeabilisation. The correlation of ultrasound-induced microbubble disruption with sonoporation efficacy is also investigated.

Section snippets

Methods

In this study, KHT-C cells were used as a biological model for investigating the sonoporation process. Cells in suspension were exposed to calibrated ultrasound beams and the effects on cell membrane permeability and viability were measured using fluorescent markers and flow cytometry. The disruption of microbubbles was characterized by measuring the size distribution of a sample of microbubbles before and after exposure.

Results

In this study, the effect of acoustic pressure, pulse centre frequency, pulse duration, pulse repetition frequency and insonation time (a total of 87 different exposure conditions) on cell permeability, cell viability and microbubble disruption was investigated. The results are shown in Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7 as mean and standard error of the mean of at least five independent measurements of cell membrane permeability and cell viability and three independent measurements

Discussion

This study demonstrates the ability of ultrasound to enhance the uptake of large molecules to which the cell membrane is normally impermeable in an in vitro cell suspension model. The results indicate that sonoporation in KHT-C cells in the presence of microbubbles depends on ultrasound exposure parameters, and that, in this experimental setting, optimisation of sonoporation is possible. This investigation established a parameter space of ultrasound- and microbubble-mediated permeabilisation of

Conclusions

Sonoporation of KHT-C cells in suspension with microbubbles was observed for a range of ultrasound exposure conditions. It was found that ultrasound causes either reversible cell membrane disruption, which in turn allows cell membrane-impermeable molecules to pass, or it causes irreversible cell membrane damage that results in cell death. Cell permeability and nonviability increase with acoustic pressure duty cycle and insonation time, and decrease with ultrasound frequency. Sonoporation was

Acknowledgments

This work was supported by grants from the Terry Fox Programme of the National Cancer Institute of Canada and the Canadian Institutes of Health Research including a Doctoral Award.

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