Tailoring the size of ultrasound responsive lipid-shelled nanodroplets by varying production parameters and environmental conditions

Liquid perfluorocarbon nanodroplets (NDs) are an attractive alternative to microbubbles (MBs) for ultrasound-mediated therapeutic and diagnostic applications. ND size and size distribution have a strong influence on their behaviour in vivo, including extravasation efficiency, circulation time, and response to ultrasound stimulation. Thus, it is desirable to identify ways to tailor the ND size and size distribution during manufacturing. In this study phospholipid-coated NDs, comprising a perfluoro-n-pentane (PFP) core stabilised by a DSPC/PEG40s (1,2-distearoyl-sn-glycero-3-phosphocholine and polyoxyethylene(40)stearate, 9:1 molar ratio) shell, were produced in phosphate-buffered saline (PBS) by sonication. The effect of the following production-related parameters on ND size was investigated: PFP concentration, power and duration of sonication, and incorporation of a lipophilic fluorescent dye. ND stability was also assessed at both 4 °C and 37 °C. When a sonication pulse of 6 s and 15% duty cycle was employed, increasing the volumetric concentration of PFP from 5% to 15% v/v in PBS resulted in an increase in ND diameter from 215.8 ± 16.8 nm to 408.9 ± 171.2 nm. An increase in the intensity of sonication from 48 to 72 W (with 10% PFP v/v in PBS) led to a decrease in ND size from 354.6 ± 127.2 nm to 315.0 ± 100.5 nm. Increasing the sonication time from 20 s to 40 s (using a pulsed sonication with 30% duty cycle) did not result in a significant change in ND size (in the range 278-314 nm); however, when it was increased to 60 s, the average ND diameter reduced to 249.7 ± 9.7 nm, which also presented a significantly lower standard deviation compared to the other experimental conditions investigated (i.e., 9.7 nm vs. > 49.4 nm). The addition of the fluorescent dye DiI at different molar ratios did not affect the ND size distribution. NDs were stable at 4 °C for up to 6 days and at 37 °C for up to 110 min; however, some evidence of ND-to-MB phase transition was observed after 40 min at 37 °C. Finally, phase transition of NDs into MBs was demonstrated using a tissue-mimicking flow phantom under therapeutic ultrasound exposure conditions (ultrasound frequency: 0.5 MHz, acoustic pressure: 2-4 MPa, and pulse repetition frequency: 100 Hz).