Intracellular vesicle entrapment of nanobubble ultrasound contrast agents targeted to PSMA promotes prolonged enhancement and stability in vivo and in vitro

Previous work has shown that active targeting of nanobubble (NB) ultrasound contrast agents to the prostate-specific membrane antigen (PSMA) significantly prolongs ultrasound signal enhancement in PSMA-expressing prostate cancer. However, the specific mechanism behind this effect is not well understood. Furthermore, prior studies were carried out using clinical ultrasound scanners in a single imaging plane. Because tumor heterogeneity can have a drastic effect on bubble kinetics and resulting contrast enhancement, a single region of interest in one imaging plane over time may not fully represent the contrast dynamics of the entire tumor. Accordingly, in the current work, we used high-frequency dynamic parametric contrast-enhanced ultrasound (DCE-US) imaging to gain a detailed understanding of NB kinetics in prostate tumors in mice. Specifically, we examined the differences in enhancement between the tumor periphery and tumor core in the same imaging plane. We also quantified intact nanobubble retention in the entire tumor volume. To better understand the mechanism behind prolonged tumor enhancement, intracellular retention and the acoustic activity of PSMA-NB were evaluated in cell culture. DCE-US US data suggest that both tumor wash-in and retention of PSMA-NB are delayed due to biomarker interaction and binding. The longer retention of PSMA-NB signal in tumor core supported target-driven bubble extravasation. In vitro studies demonstrated a higher level of internalization and prolonged-acoustic activity of internalized PSMA-NB. GC/MS analysis confirmed gas persistence in the cells after PSMA-NB internalization. The active-targeting of NB results in cellular internalization via receptor-mediated endocytosis, and the location with intracellular vesicles (late-stage endosomes/lysosomes) significantly prolongs gas retention within the cells. These features can enable background-free diagnostic imaging of the target cells/tissues, as well as highly focused ultrasound-modulated therapeutic interventions. ### Competing Interest Statement The authors have declared no competing interest.