Ultrasound-triggered release of an active Wnt signalling agent (6-bromoindirubin-3-oxime) from perfluorocarbon nanodroplets.

Ultrasound-activated perfluorocarbon nanodroplets (NDs) have potential as circulating agents for non-invasive, targeted drug delivery. NDs can be induced to cavitate under focussed ultrasound stimulation, releasing a therapeutic payload. They have been explored widely as a means of drug delivery in oncology but there is less information available on their use in regenerative medicine. Here, we tested the hypothesis that NDs could be fabricated to incorporate an agonist of Wnt signalling, a pathway common to many regenerative tissue-specific processes, and to release it in active form upon ultrasound stimulation. NDs were formed by ultrasonic emulsification of perfluorobutane in the presence of 6-bromoindirubin-3-oxime (BIO) and phospholipids: DSPC and DSPE-PEG2000. The nanodroplet formulation (∼200 nm diameter and 5 × 109 mL-1) was stable in aqueous suspension for up to 10 days at 4 °C, but gradual passive release of BIO was observed over a period of hours (15-30% release in 24 hours) in serum-containing growth medium at 37 °C, as measured by high pressure liquid chromatography. NDs were not toxic to either primary or continuous bone-derived cells and induced signalling in a 3T3 Wnt reporter cell line, indicating that the released BIO was active. Rapid release of BIO was achieved by exposing NDs to ultrasound. 25% BIO release was observed after 30 s of exposure to ultrasound (0.9 MHz centre frequency, 0.75 MPa peak negative pressure, 1 kHz pulse repetition frequency, 10% duty cycle) and activated Wnt signalling in cells. Our data indicate that NDs may be used to incorporate and deliver a Wnt pathway activator in response to ultrasound stimulation and that they have potential as drug delivery agents for regenerative medicine applications.