Abstract
BACKGROUND: Sonodynamic therapy (SDT) is a promising non-invasive modality for cancer treatment. This in vitro study aims to investigate the cytotoxic effect of low-intensity ultrasound (US) combined with microbubbles (MBs)-enhanced 5-aminolevulinic acid (5-ALA)-mediated SDT on C6 glioma cells. METHODS: In vitro experiments utilized the C6 glioma cell line. Cultured cells were subjected to low-intensity US irradiation [1.0 MHz, acoustic intensity of 2.0 W/cm(2), peak negative pressure of 0.5 MPa, mechanical index (MI) of 0.5, duty ratio of 20%] after the administration of 5-ALA. The uptake of 5-ALA by the cells was assessed using confocal laser scanning microscope (CLSM) and flow cytometry. Cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Transmission electron microscopy (TEM) was employed to observe the ultrastructure and mitochondrial changes in the cells. The generation of reactive oxygen species (ROS) within the cells was examined using confocal CLSM. RESULTS: Flow cytometry and CLSM results indicated that the level of protoporphyrin IX (PpIX) in cells was significantly enhanced after treatment with US combined with MBs and 5-ALA (US-MBs-5-ALA group). MTS analysis demonstrated that the cell viability was significantly reduced in the US-MBs-5-ALA group. Qualitative observations from TEM revealed that mitochondria in cells subjected to low-intensity US treatment alone showed swelling and vacuolization. Upon combined treatment with US and 5-ALA, cells exhibited apoptotic features such as nuclear condensation and crescent-shaped structures. Moreover, mitochondrial damage was more severe in the US-MBs-5-ALA group. Qualitative analysis by CLSM showed that the level of ROS in the US-MBs-5-ALA group was significantly increased. These results suggest a mechanistic link where US-MBs enhance 5-ALA uptake, leading to increased ROS generation under sonication, which in turn induces mitochondrial damage and promotes cytotoxicity. CONCLUSIONS: These preliminary in vitro findings demonstrate that low-intensity US combined with MBs can increase the uptake of 5-ALA by C6 cells and enhance the cytotoxic effect of 5-ALA-mediated SDT. This approach warrants further investigation as a potential therapeutic strategy for glioma.