Ultrasound-activated nanobubbles are being widely investigated as contrast agents and therapeutic vehicles. Nanobubbles hold potential for accessing the tumor extravascular compartment, though this relies on clinically debated passive accumulation for which evidence to date is indirect. We recently reported ultrasound-triggered conversion of high payload porphyrin-encapsulated microbubbles to nanobubbles, with actively enhanced permeability for local delivery. This platform holds implications for optical/ultrasound-based imaging and therapeutics. While promising, it remains to be established how nanobubbles are generated and whether they extravasate intact. Here, insights into the conversion process are reported, complemented by novel simultaneous intravital and acoustic monitoring in tumor-affected functional circulation. The first direct acoustic evidence of extravascular intact nanobubbles are presented. These insights collectively advance this delivery platform and multimodal micro- and nanobubbles, extending their utility for imaging and therapeutics within and beyond the vasculature.

Background: There has been growing interest in nanobubbles for their potential to extend bubble-mediated ultrasound approaches beyond that of their larger microbubble counterparts. In particular, the smaller scale of nanobubbles may enable them to access the tumor extravascular compartment for imaging and therapy in closer proximity to cancer cells. Compelling preliminary demonstrations of the imaging and therapeutic abilities of nanobubbles have thus emerged, with emphasis on their ability to extravasate. However, studies to date rely on indirect histologic evidence that cannot confirm whether the structures remain intact beyond the vasculature - leaving their extravascular potential largely untapped. Methods: Nanobubble acoustic scattering was assessed using a recently reported ultra-stable formulation at low concentration (10 6 mL -1 ) and frequency (1 MHz), over a range of pressures (100-1500 kPa) in a channel phantom. The pressure-dependent response was utilized as a basis for in vivo experiments where ultrasound transmitters and receivers were integrated into a window chamber for simultaneous intravital multiphoton microscopy and acoustic monitoring in tumor-affected microcirculation. Microscopy and acoustic data were utilized to assess passive and active delivery of nanobubbles and determine whether they remained intact beyond the vasculature. Results: Nanobubbles exhibit pressure-dependent nonlinear acoustic scattering. Nanobubbles are also found to have prolonged acoustic vascular pharmacokinetics, and passively extravasate intact into tumors. Ultrasound stimulation of nanobubbles is shown to actively enhance the delivery of both intact nanobubbles and shell material, increasing their spatial bioavailability deeper into the extravascular space. A range of acute vascular effects were also observed. Conclusion: This study presents the first direct evidence that nanobubbles passively and actively extravasate intact in tumor tissue, and is the first to directly capture acute vascular events from ultrasound-stimulation of nanobubbles. The insights gained here demonstrate an important step towards unlocking the potential of nanobubbles and extending ultrasound-based applications.