A comparative analysis of low intensity ultrasound effects on living cells: from simulation to experiments

Abstract

Ultrasounds are already broadly exploited in clinical diagnostics and are now becoming a powerful and not harmful tool in antitumoral therapies, as they are able to produce damages towards cancer cells, thank to inertial cavitation and temperature increase. The use of US alone or combined to molecular compounds, microbubbles or solid-state nanoparticles is the focus of current research and clinical trials, like thermoablation, drug sonoporation or sonodynamic therapies. In the present work, we discuss on the non-thermal effects of ultrasound and the conditions which enable oxygen radical production and which role they can have in provoking the death of different cancer cell lines. In this perspective, we set a mathematical model to predict the pressure spatial distribution in a defined water sample volume and thus obtain a map of acoustic pressures and acoustic intensities of the applied ultrasound at different input powers. We then validate and verify these numerical results with direct acoustic measurements and by detecting the production of reactive oxygen species (ROS) by means of sonochemiluminescence (SCL) and electron paramagnetic resonance (EPR) spectroscopy, applied to the same water sample volume and using the same US input parameters adopted in the simulation. Finally, the various US conditions are applied to two different set of cancer cell lines, a cervical adenocarcinoma and a hematological cancer, Burkitt's lymphoma. We hypothesize how the ROS generation can influence the recorded cell death. In a second set of experiments, the role of semiconductor metal oxide nanocrystals, i.e. zinc oxide, is also evaluated by adding them to the water and biological systems. In particular, the role of ZnO in enhancing the ROS production is verified. Furthermore, the interplay among US and ZnO nanocrystals is evaluated in provoking cancer cell death at specific conditions. This study demonstrates a useful correlation between numerical simulation and experimental acoustic validation as well as with ROS measurement at both qualitative and quantitative levels during US irradiation of simple water solution. It further tries to translate the obtained results to justify one of the possible mechanisms responsible of cancer cell death. It thus aims to pave the way for the use of US in cancer therapy and a better understanding on the non-thermal effect that a specific set of US parameters can have on cancer cells cultured in vitro.

Keywords: Acoustic cavitation; Acoustic field simulation; Electron paramagnetic spectroscopy; Nanoparticles; Reactive oxygen species; Sonochemiluminescence.

Fig. 1

Scheme of the experimental set-up (left) and simulated domain (right), whose description and characteristic dimensions are reported in Table 1

Fig. 2

Acquired image from the 24-well plate and its processing. The image resulting from the sonication A is subjected to subtraction of the background image and the selection of a specific circular ROI B. These images refer to the particular sonication condition with US power = 0.6 W/cm² and sonication time 3 min

Fig. 3

Acoustic pressure field (top panels) and acoustic density distributions (bottom panels) within the well simulated with COMSOL^® Multiphysics for I_input = 0.45 W/cm², along vertical (A and C) and horizontal (B and D) planes of the sample well containing water. The darkest red and blue colors in panels A and B indicate the maximum and the minimum values, respectively, that the acoustic pressure can assume, equal to + 0.52 MPa and—0.55 MPa. The darkest blue and red colors of the acoustic density (panels C and D) correspond to 0 W/cm² and 4.96 W/cm², respectively

Fig. 4

The generation of ROS in bd water volumes and the possible effects on living cell cultures: (A) Spatial average blue intensity of luminol due to sonochemiluminescence (SCL); (B) Molar concentration of DMPO-OH spin adducts obtained during EPR measurements; (C) KB cancer cell viability and (D) Daudi cell viability after 24 h from US irradiation. In these experiments, UT means untreated cells, and the presence of ZnO-NH₂ NCs is not considered

Fig. 5

Overview of the ROS generation and related cell viability in a system comprising inorganic metal oxide nanoparticles (ZnO-NH₂ NCs) and ultrasound irradiation in a well from a 24-multiwell plate. (A) Luminol blue intensity from SCL measurements with 200 μg/ml of ZnO-NH₂ NCs; (B) DMPO-OH adduct concentration measurements from EPR spectroscopy to evaluate ROS production (with 200 μg/ml of ZnO-NH₂ NCs), and (C) KB cancer cell viability according to different US power densities and sonication times (with 24 h preincubation of cell with 10 μg/ml of ZnO-NH₂ NCs); black bar and red dotted bar correspond respectively to untreated KB cell and cells incubated with NCs without US exposure