RATIONALE: Lipid-shelled nanobubbles (NBs) can be visualized and activated using noninvasive ultrasound (US) stimulation, leading to significant bioeffects. We have previously shown that active targeting of NBs to prostate-specific membrane antigen (PSMA) overexpressed in prostate cancer (PCa) enhances the cellular internalization and prolongs retention of NBs with persistent acoustic activity (~hrs.). In this work, we hypothesized that tumor-accumulated PSMA-NBs combined with low frequency therapeutic US (TUS) will lead to selective damage and induce a therapeutic effect in PSMA-expressing tumors compared to PSMA-negative tumors. METHODS: PSMA-targeted NBs were formulated by following our previously established protocol. Cellular internalization of fluorescent PSMA-NBs was evaluated by confocal imaging using late endosome/lysosome staining pre- and post-TUS application. Two animal models were used to assess the technique. Mice with dual tumors (PSMA expressing and PSMA negative) received PSMA-NB injection via the tail vein followed by TUS 1 hr. post injection (termed, targeted NB therapy or TNT). Twenty-four hours after treatment mice were euthanized and tumor cell apoptosis evaluated via TUNEL staining. Mice with single tumors (either PSMA + or -) were used for survival studies. Tumor size was measured for 80 days after four consecutive TNT treatments (every 3 days). To test the approach in a larger model, immunosuppressed rabbits with orthotopic human PSMA expressing tumors received PSMA-NB injection via the tail vein followed by TUS 30 min after injection. Tumor progression was assessed via US imaging and at the end point apoptosis was measured via TUNEL staining. RESULTS: In vitro TNT studies using confocal microscopy showed that the internalized NBs and cellular compartments were disrupted after the TUS application, yet treated cells remained intact and viable. In vivo, PSMA-expressing tumors in mice receiving TNT treatment demonstrated a significantly greater extent of apoptosis (78.45 ± 9.3%, p < 0.01) compared to the other groups. TNT treatment significantly inhibited the PSMA (+) tumor growth and overall survival significantly improved (median survival time increase by 103%, p < 0.001). A significant reduction in tumor progression compared to untreated control was also seen in the rabbit model in intraprostatic (90%) and in extraprostatic lesions (94%) (p = 0.069 and 0.003, respectively). CONCLUSION: We demonstrate for the first time the effect of PSMA-targeted nanobubble intracellular cavitation on cancer cell viability and tumor progression in two animal models. Data demonstrate that the targeted nanobubble therapy (TNT) approach relies primarily on mechanical disruption of intracellular vesicles and the resulting bioeffects appear to be more specific to target cancer cells expressing the PSMA receptor. The effect, while not lethal , resulted in significant tumor apoptosis in both a mouse and a rabbit model of PCa. While the mechanism of action of these effects is yet unclear, it is likely related to a locally-induced immune response, opening the door to future investigations in this area.