Exploiting ultrasound-mediated effects in delivering targeted,site-specific cancer therapy

Although the concept of employing ultrasound for the treatment of cancer is not a new one, virtually all existing ultrasound-based clinical cancer treatments are based on hyperthermic ablation. This review seeks to highlight the potential offered by more subtle ultrasound-triggered phenomena such as sonoporation in delivering novel targeted cancer treatment modalities.     

Examination of inertial cavitation of Optison in producing sonoporation of chinese hamster ovary cells

The objective of this project was to elucidate the relationship between ultrasound contrast agents (UCAs) and sonoporation. Sonoporation is an ultrasound-induced, transient cell membrane permeability change that allows for the uptake of normally impermeable macromolecules. Specifically, this study will determine the role that inertial cavitation plays in eliciting sonoporation. The inertial cavitation thresholds of the UCA, Optison, are compared directly with the results of sonoporation to determine the involvement of inertial cavitation in sonoporation. Chinese hamster ovary (CHO) cells were exposed as a monolayer in a solution of Optison, 500,000 Da fluorescein isothiocyanate-dextran (FITC-dextran), and phosphate-buffered saline (PBS) to 30 s of pulsed ultrasound at 3.15-MHz center frequency, 5-cycle pulse duration and 10-Hz pulse repetition frequency. The peak rarefactional pressure (P(r)) was varied over a range from 120 kPa-3.5 MPa, and five independent replicates were performed at each pressure. As the P(r) was increased, from 120 kPa-3.5 MPa, the fraction of sonoporated cells among the total viable population increased from 0.63-10.21%, with the maximum occurring at 2.4 MPa. The inertial cavitation threshold for Optison at these exposure conditions has previously been shown to be in the range 0.77-0.83 MPa, at which sonoporation activity was found to be 50% of its maximum level. Furthermore, significant sonoporation activity was observed at pressure levels below the threshold for inertial cavitation of Optison. Above 2.4 MPa, a significant drop in sonoporation activity occurred, corresponding to pressures where >95% of the Optison was collapsing. These results demonstrate that sonoporation is not directly a result of inertial cavitation of the UCA, rather that the effect is related to linear and/or nonlinear oscillation of the UCA occurring at pressure levels below the inertial cavitation threshold.     

Controlled permeation of cell membrane by single bubble acoustic cavitation

Sonoporation is the membrane disruption generated by ultrasound and has been exploited as a non-viral strategy for drug and gene delivery. Acoustic cavitation of microbubbles has been recognized to play an important role in sonoporation. However, due to the lack of adequate techniques for precise control of cavitation activities and real-time assessment of the resulting sub-micron process of sonoporation, limited knowledge has been available regarding the detail processes and correlation of cavitation with membrane disruption at the single cell level. In the current study, we developed a combined approach including optical, acoustical, and electrophysiological techniques to enable synchronized manipulation, imaging, and measurement of cavitation of single bubbles and the resulting cell membrane disruption in real-time. Using a self-focused femtosecond laser and high frequency ultrasound (7.44 MHz) pulses, a single microbubble was generated and positioned at a desired distance from the membrane of a Xenopus oocyte. Cavitation of the bubble was achieved by applying a low frequency (1.5 MHz) ultrasound pulse (duration 13.3 or 40 μs) to induce bubble collapse. Disruption of the cell membrane was assessed by the increase in the transmembrane current (TMC) of the cell under voltage clamp. Simultaneous high-speed bright field imaging of cavitation and measurements of the TMC were obtained to correlate the ultrasound-generated bubble activities with the cell membrane poration. The change in membrane permeability was directly associated with the formation of a sub-micrometer pore from a local membrane rupture generated by bubble collapse or bubble compression depending on ultrasound amplitude and duration. The impact of the bubble collapse on membrane permeation decreased rapidly with increasing distance (D) between the bubble (diameter d) and the cell membrane. The effective range of cavitation impact on membrane poration was determined to be D/d = 0.75. The maximum mean radius of the pores was estimated from the measured TMC to be 0.106 ± 0.032 μm (n = 70) for acoustic pressure of 1.5 MPa (duration 13.3 μs), and increased to 0.171 ± 0.030 μm (n = 125) for acoustic pressure of 1.7 MPa and to 0.182 ± 0.052 μm (n = 112) for a pulse duration of 40 μs (1.5 MPa). These results from controlled cell membrane permeation by cavitation of single bubbles revealed insights and key factors affecting sonoporation at the single cell level.     

In Vitro Gene Transfer by Electrosonoporation

Among the nonviral methods for gene delivery in vitro, electroporation is simple, inexpensive and safe. To upregulate the expression level of transfected gene, we investigated the applicability of electrosonoporation. This approach consists of a combination of electric pulses and ultrasound assisted with gas microbubbles. Cells were first electroporated with plasmid DNA encoding–enhanced green fluorescent protein and then sonoporated in presence of contrast microbubbles. Twenty-four hours later, cells that received electrosonoporation demonstrated a four-fold increase in transfection level and a six-fold increase in transfection efficiency compared with cells having undergone electroporation alone. Although electroporation induced the formation of DNA aggregates into the cell membrane, sonoporation induced its direct propulsion into the cytoplasm. Sonoporation can improve the transfer of electro-induced DNA aggregates by allowing its free and rapid entrance into the cells. These results demonstrated that in vitro gene transfer by electrosonoporation could provide a new potent method for gene transfer. (E-mail: rols@ipbs.fr)     

Optimising ultrasound-mediated gene transfer (sonoporation) in vitro and prolonged expression of a transgene in vivo: potential applications for gene therapy of cancer

Therapeutic approaches using gene-based medicines promise alternatives or adjuncts to conventional cancer treatment. Because of its non-invasive nature, ultrasound, as a membrane-permeabilising stimulus has the potential to be highly competitive with viral gene delivery and existing non-viral alternatives. In optimising ultrasound-mediated, microbubble-assisted (MB101) gene tranfection in vitro, we demonstrate efficiencies of up to 18% using ultrasound at 1 MHz at a duty cycle of 25% at intensities ranging from 1 to 4 W cm(-2). Using ultrasound-mediated transfection together with an episomal plasmid-based gene expression system, we demonstrate prolonged functional gene expression of luciferase in mouse hind leg muscle and in tumours in vivo.     

Ultrasound-enhanced drug dispersion through solid tumours and its possible role in aiding ultrasound-targeted cancer chemotherapy

It has been demonstrated that inadequate dispersion of cancer chemotherapeutic drugs throughout the tissues of larger, relatively poorly vascularised tumours compromises the therapeutic effectiveness of such drugs. Recently we demonstrated that electric fields could be exploited to achieve dispersion of a cancer chemotherapeutic drug through relatively impermeable tissues of a poorly vascularised solid tumour model. Using a modified Sonidel SP100 sonoporator we demonstrate that ultrasound may enhance the toxicity of a cancer chemotherapeutic drug by dispersing the drug through relatively impermeable tissues of a non-vascularised tumour model in vivo. We suggest that such a phenomenon may play a significant role in ultrasound targeting of cancer chemotherapeutic drugs, particularly in the treatment of solid tumours.     

Spatiotemporal Effects of Sonoporation Measured by Real-Time Calcium Imaging

To investigate the effects of sonoporation, spatiotemporal evolution of ultrasound-induced changes in intracellular calcium ion concentration ([Ca²⁺]_i) was determined using real-time fura-2AM fluorescence imaging. Monolayers of Chinese hamster ovary (CHO) cells were exposed to a 1-MHz ultrasound tone burst (0.2 s, 0.45 MPa) in the presence of Optison^™ microbubbles. At extracellular [Ca²⁺]_o of 0.9 mM, ultrasound application generated both nonoscillating and oscillating (periods 12 to 30 s) transients (changes of [Ca²⁺]_i in time) with durations of 100–180 s. Immediate [Ca²⁺]_i transients after ultrasound application were induced by ultrasound-mediated microbubble–cell interactions. In some cases, the immediately affected cells did not return to pre-ultrasound equilibrium [Ca²⁺]_i levels, thereby indicating irreversible membrane damage. Spatial evolution of [Ca²⁺]_i in different cells formed a calcium wave that was observed to propagate outward from the immediately affected cells at 7–20 μm/s over a distance >200 μm, causing delayed transients in cells to occur sometimes 60 s or more after ultrasound application. In calcium-free solution, ultrasound-affected cells did not recover, consistent with the requirement of extracellular Ca²⁺ for cell membrane recovery subsequent to sonoporation. In summary, ultrasound application in the presence of Optison^™ microbubbles can generate transient [Ca²⁺]_i changes and oscillations at a focal site and in surrounding cells via calcium waves that last longer than the ultrasound duration and spread beyond the focal site. These results demonstrate the complexity of downstream effects of sonoporation beyond the initial pore formation and subsequent diffusion-related transport through the cellular membrane. (E-mail address: cxdeng@umich.edu)     

Quantitative relations of acoustic inertial cavitation with sonoporation and cell viability

Ultrasound-induced acoustic cavitation assists gene delivery, possibly by increasing the permeability of the cell membranes. How the cavitation dose is related to the sonoporation rate and the cell viability is still unknown and so this in vitro study quantitatively investigated the effects of cavitation induced by 1-MHz pulsed ultrasound waves and the contrast agent Levovist® (containing microbubbles when reconstituted by adding saline and shaken) on the delivery of short DNA-FITC molecules into HeLa cells. The concentrations of cells and DNA-FITC were 2 × 10⁵ cells/mL and 40 μg/mL, respectively. The cavitation was quantified as the inertial cavitation dose (ICD), corresponding to the spectral broadband signal enhancement during microbubble destruction. The relations of ICD with sonoporation and cell viability were examined for various acoustic pressures (0.48–1.32 MPa), Levovist® concentrations (1.12 × 10⁵−1.12 × 10⁷ bubbles/mL) and pulse durations (1–10 cycles). The linear regressions of the sonoporation rate versus ICD and the cell viability versus ICD were y = 28.67x + 10.71 (R² = 0.95) and z = −62.83x + 91.18 (R² = 0.84), respectively, where x is ICD, y is the sonoporation rate and z is the cell viability. These results show that the sonoporation rate and the cell viability are highly correlated with the ICD, indicating that sonoporation results may be potentially predicted using ICD. (E-mail: paichi@cc.ee.ntu.edu.tw)