Nanobubble-mediated sonodynamic therapy enhances cuproptosis in the treatment of hepatocellular carcinoma

<i>Background and objective</i>: Hepatocellular carcinoma (HCC) is one of the primary causes of cancer-associated death worldwide, which has few viable therapeutic options at later stages. Cuproptosis, a newly discovered kind of programmed cell death, gives a new therapeutic target for HCC. Nanobubble (NB)-mediated sonodynamic therapy (SDT) has been a prospective method for augmenting the efficacy and delivery of therapeutic agents. This study investigates the potential of NB-mediated SDT to enhance cuproptosis in HCC treatment, aiming to evaluate the efficacy of NB-mediated SDT in enhancing cuproptosis in HCC cells and to clarify the potential mechanisms. <i>Methods</i>: NBs were engineered to encapsulate the sonosensitizer protoporphyrin IX (PPIX) and the cuproptosis inducer elesclomol-Cu²⁺ (ES-Cu), forming PPIX@ES-Cu NBs, a targeted delivery system activated by ultrasound. HCC cell lines (Hepa1-6) were treated with these PPIX@ES-Cu NBs followed by low-intensity ultrasound (LIUS) exposure. Mitochondrial function and cell viability were evaluated through CCK assays and fluorescence microscopy. qPCR was implemented for examining the expression of cuproptosis-associated genes. <i>Results</i>: The NB-mediated SDT significantly enhanced the delivery of copper ions and sonosensitizers to HCC cells. Upon ultrasound activation, there was a substantial increase in ROS production and intracellular copper accumulation. This led to mitochondrial dysfunction, characterized by decreased mitochondrial membrane potential and disrupted tricarboxylic acid (TCA) cycle enzyme activities. Treated cells exhibited increased expression of cuproptosis markers, including upregulation of lipoylated mitochondrial proteins and proteotoxic stress indicators. Cell viability assays demonstrated a considerable decrease in HCC cell survival compared to controls. <i>Conclusion</i>: NB-mediated SDT effectively enhances cuproptosis in HCC cells by facilitating targeted delivery and activation of therapeutic agents. The synergistic effect of ROS generation and copper accumulation induces cuproptosis, offering a novel and potent therapeutic strategy for HCC treatment. These results lay the groundwork for more <i>in vivo</i> research and possible clinical use of this combo treatment.