LOW-FREQUENCY SONODYNAMIC THERAPY: CAVITATION ENHANCED BY GAS VESICLES FROM HALOBACTERIUM SALINARUM

Sonodynamic therapy (SDT) relies on ultrasound-induced acoustic cavitation to activate sonosensitizers and promote the generation of reactive oxygen species (ROS). Gas-filled microbubbles are widely used as ultrasound contrast agents for diagnostic imaging; however, their micrometer-scale size may limit extravasation and accumulation in solid tumors. Nanoscale gas bubbles could overcome this limitation, potentially enabling their use not only for imaging but also as cavitation nuclei for therapeutic applications. Gas vesicles (GVs) are genetically encoded, protein-shelled nanostructures produced by microorganisms that contain gas and can interact with ultrasound, making them promising nanoscale cavitation agents. Their response to ultrasound could potentially be exploited at lower intensities for imaging and at higher intensities to promote cavitation-mediated SDT. Therefore, this study aimed to obtain GVs from Halobacterium salinarum, characterize their physicochemical properties, and evaluate their ability to modulate cavitation induced by low-frequency ultrasound (LFU, 20 kHz). The H. salinarum NRC-1 strain was cultured in CM medium for approximately seven days until reaching an optical density at 500 nm (OD₅₀₀) of approximately 2.0. GVs were isolated by cell lysis, followed by centrifugation at 300 × g for 4 h and washing with PBS. Their hydrodynamic diameter and zeta potential were determined, while morphology was evaluated by cryogenic transmission electron microscopy (cryo-TEM). Cavitation modulation was assessed using the indirect potassium iodide method and confirmed by high-speed imaging. GVs exhibited a mean hydrodynamic diameter of 196±8 nm, a polydispersity index of 0.03±0.01, and a zeta potential of −27±4 mV, indicating a highly homogeneous size distribution. Cryo-TEM revealed predominantly biconical morphology. At OD₅₀₀ = 0.3, GV-containing suspensions exhibited approximately 30-fold greater cavitation activity than pure water under identical conditions. High-speed imaging revealed repeated nucleation, growth, and collapse of cavitation bubbles exclusively in GV-containing suspensions, with bubbles reaching approximately 140 μm in diameter. Overall, GVs effectively enhanced LFU-induced cavitation, supporting their potential as nanoscale cavitation agents for diagnostic imaging and SDT.