Systematic Analysis of Ultrasound Parameters for Gene Delivery Efficiency and Cell Viability in 4T1 Cells

Gene therapy is a promising strategy for treating various diseases. However, its application remains limited by inefficient intracellular delivery, particularly in difficult-to-transfect cells such as triple-negative breast cancer (TNBC). Ultrasound-mediated gene delivery has emerged as a non-invasive approach to enhance membrane permeability, but its efficiency and safety depend strongly on acoustic parameters. In this study, we systematically investigated the effects of ultrasound intensity, pulse repetition frequency (PRF), duty cycle, and exposure time on gene delivery efficiency and cytotoxicity in 4T1 murine TNBC cells using microbubble-mediated sonoporation. Gene expressions were evaluated using a luciferase assay, and cell viability was assessed using an MTT assay. Total acoustic energy was calculated to analyze the relationship between cumulative ultrasound exposure and biological responses. Increasing total acoustic energy was associated with enhanced gene expression and reduced cell viability. However, gene expression increased only up to a certain level and then showed no further substantial increase, whereas cell viability continued to decline. Parameter-specific analysis further indicated that biological responses were not determined by total acoustic energy alone. These findings suggest that both cumulative acoustic energy and individual ultrasound parameters should be considered when optimizing ultrasound-mediated gene delivery to balance transfection efficiency and cytotoxicity.