Extended Data Fig. 1: Acoustic characterization of uncaging and in vivo assessment of cavitation risk.
a. Schematic of flow chamber with thin-walled silicone tubing (0.1 mm wall thickness, 18 gauge) submerged in a large temperature controlled degassed water bath kept at 37 °C. The focal zone of the focused ultrasound transducer was placed on the tubing and a hydrophone placed at 2.5 cm from the tubing, with 90° between the hydrophone and the transducer, recording acoustic backscatter and emissions during sonication of infusion of one of four media: degassed saline (dilution medium used in vitro experiments), the liposome internal medium (250 mM NH4(SO4)2 + 5% sucrose), the S5-KL = SonoKet liposomes diluted in saline to its estimated in vivo circulating concentration based on the observed in vivo ketamine blood concentrations (~109/mL particle concentration) and commercial microbubbles diluted to its estimated in vivo circulating concentration during blood-brain barrier opening experiments (~108/mL particle concentration). (https://biorender.com/i9qghda) b. Ketamine uncaging with 250 or 650 kHz sonication. Peak negative pressures of 0.7-1.1 MPa and 1.7-2.8 MPa for 250 and 650 kHz respectively (left), providing matching mechanical indices of 1.4-2.2 (right). Dotted line: drug release without FUS in this apparatus. Data presented as mean and individual data points (n = 2). c. Received echo spectral magnitude following sonication of saline (Black), gas-filled microbubbles (Purple), liposome internal medium (Pink), or liposomes (S5-KL; Teal) versus frequency with 0.3, 0.9, 1.1 and 1.5 MPa in situ peak negative pressure, 250 kHz center frequency, 1 ms duration. d. To assess in vivo cavitation effects, S5-KL or commercial microbubbles were co-administered i.v. with Evans Blue dye and FUS was applied transcranially to the posterior right cortex of rats (center frequency 250 kHz, peak negative pressure of 1.1 MPa and 25% duty cycle for S5-KL, peak negative pressure of 0.5 MPa and 1% duty cycle for microbubbles). Brain dye extravasation was readily observed with microbubbles, indicating in vivo blood-brain barrier opening due to cavitation, with no such dye extravasation noted with S5-KL under gross or microscopic analysis of tissue sections, indicating no observable bioeffects of potential cavitation with S5-KL under these conditions. e. Representative TEM images of different API loaded liposomes show spherical morphology, with a liquid core with no observable gas or voids. (Scale Bar=100 nm).
