Gas-filled protein nanostructures as cavitation nuclei for molecule-specific sonodynamic therapy

Sonodynamic therapy (SDT) is a promising alternative for cancer therapy, understood to exert cytotoxicity through cavitation and subsequent production of large amounts of reactive oxygen species (ROS). Gas-filled protein nanostructures (gas vesicles or GVs) produced by cyanobacteria have a hollow structure similar to microbubbles and have demonstrated comparable enhancement of ultrasound imaging contrast. We thus hypothesized that GVs may act as stable nuclei for inertial cavitation to enhance SDT with improved enhanced permeability and retention (EPR) effects due to their nanometer scale. The function of GVs to mediate cavitation, ROS production, and cell-targeted toxicity under SDT was determined. In solution, we found that GVs successfully increased cavitation and enhanced ROS production in a dose- and time-dependent manner. Then, GV surfaces were modified (FGVs) to specifically target CD44⁺ cells and accumulate preferentially at the tumor site. In vitro sonodynamic therapy (SDT) showed ROS production and tumor cell toxicity substantially elevated in the presence of FGVs, and the addition of FGVs was found to enhance cavitation and subsequently inhibit tumor growth and exert greater damage to tumors under SDT in vivo. Our results thus demonstrate that FGVs can function as stable, nanosized, nuclei for spatially accurate and cell-targeted SDT. STATEMENT OF SIGNIFICANCE: The initiation of inertial cavitation is critical for ROS generation and subsequent cellular toxicity in SDT. Thus, precise control of the occurrence of cavitation is a key factor in increasing SDT's therapeutic efficacy. We explored nanometer-sized gas vesicles (GVs) as a new class of cavitation nuclei for molecule-specific sonodynamic therapy. Our results showed that GV-mediated SDT treatment enabled targeted disruption of specific cells expressing a known surface marker within the area of insonation, providing a spatially specific and targeted SDT treatment.