DEVELOPMENT AND VALIDATION OF AN IN VITRO ULTRASOUND IRRADIATION PLATFORM FOR SONODYNAMIC THERAPY APPLICATIONS

Sonodynamic therapy (SDT) has emerged as a promising non-invasive approach for various therapeutic applications, including cancer treatment and microbial inactivation. This technique employs low-intensity ultrasound in combination with ultrasound-activated sensitizing agents and molecular oxygen. The combination of these components leads to the generation of reactive oxygen species (ROS) via acoustic cavitation in the biological environment, inducing cytotoxic effects on target cells. In in vitro SDT studies, various experimental configurations for ultrasound irradiation have been employed; however, the rationale for selecting a specific configuration, as well as its validation for experimental use, is seldom described in the literature. The objective of this study was to develop a platform for conducting in vitro SDT experiments in multi-well cell plates and to validate it through cavitation monitoring using potassium iodide (KI). The platform consists of a custom-designed glass tank (17.5x17x13cm) with a bottom inlet for attaching an ultrasound transducer head and a vertically adjustable plate holder to vary the distance between the transducer and the plate. During validation tests, the tank was filled with degassed water, prepared by boiling deionized distilled water, cooling it to 30°C, and sonicating to remove residual bubbles. A 96-well plate containing 0.1M KI in each well was positioned in contact with the water surface at different heights. Ultrasound was applied at a frequency of 1MHz and an intensity of 2 mW/cm² in pulsed mode (20% duty cycle) for 30 and 60 minutes. Five heights (5, 10, 15, 16 and 17cm) were tested considering the near-field distance to evaluate energy distribution across the wells. Subsequently, the absorbance was measured at 352nm with a microplate reader. The energy distribution exhibited a circular pattern (≈3 cm radius), with maximum intensity and homogeneity at a height of 16cm, localized within the six central wells. These results were consistent with previous ultrasound propagation profile characterization and mapping through sonometry using a hydrophone-based scanning tank. This work demonstrates that cavitation mapping using KI is a simplified and reproducible method for assessing ultrasound delivery in multi-well plates, providing an approach that can enhance the reliability and comparability of in vitro SDT studies.