Ultrasound contrast agents, comprised of phospholipid-coated microbubbles, can be produced as monodisperse populations using a microfluidic flow-focusing device. However, microbubble coalescence remains a significant challenge. High production temperatures (e.g., 55 °C) can be used to suppress coalescence, but it complicates the microfluidic device design and is incompatible with targeting agents and drug conjugates. This study investigates the production of monodisperse microbubbles at room temperature with the addition of the amphiphilic surfactant Pluronic F68. Two 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-based phospholipid formulations were investigated: F1, containing 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carbonyl-methoxypolyethylene glycol] (DPPE-PEG5000), and F2, which included both DPPE-PEG5000 and polyoxyethylene(40) stearate (PEG40-stearate). We characterized the size stability and acoustic behavior of monodisperse microbubbles produced with various Pluronic F68 concentrations. Adding 5-10 mol % Pluronic F68 was found to effectively suppress coalescence and facilitated the production of monodisperse microbubbles that remained shelf stable for at least 7 days. Acoustic attenuation measurements revealed a shell stiffness ranging from 0.78 to 0.93 N/m for these microbubbles. The 10 mol % Pluronic F68 addition (10PF) demonstrated superior monodispersity and was selected for further experiments. Upon dilution, the size and resonance frequencies of both F1-10PF and F2-10PF decreased over time, though F2-10PF showed better stability compared to F1-10PF for both metrics. Both F1-10PF and F2-10PF exhibited a stronger subharmonic scattering intensity than SonoVue (clinical approved microbubbles), which offers potential for blood pressure sensing. Our study shows that incorporating Pluronic F68 facilitates the production of monodisperse microbubbles at room temperature that are stable long-term and have excellent acoustical properties, with the F2-10PF formulation demonstrating better stability than the F1-10PF. ... Read more

Drug transport from blood to extravascular tissue can locally be achieved by increasing the vascular permeability through ultrasound-activated microbubbles. However, the mechanism remains unknown, including whether short and long cycles of ultrasound induce the same onset rate, spatial distribution, and amount of vascular permeability increase. Accurate models are necessary for insights into the mechanism so a microvessel-on-a-chip is developed with a membrane-free extravascular space. Using these microvessels-on-a-chip, distinct differences between 2 MHz ultrasound treatments are shown with 10 or 1000 cycles. The onset rate is slower for 10 than 1000 cycles, while both cycle lengths increase the permeability in spot-wise patterns without affecting microvessel viability. Significantly less vascular permeability increase and sonoporation are induced for 10 versus 1000 cycles at 750 kPa (i.e., the highest studied peak negative acoustic pressure (PNP)). The PNP threshold for vascular permeability increases is 750 versus 550 kPa for 10 versus 1000 cycles, while this is 750 versus 220 kPa for sonoporation. Vascular permeability increases do not correlate with αvβ3-targeted microbubble behavior, while sonoporation correlates with αvβ3-targeted microbubble clustering. In conclusion, the further mechanistic unraveling of vascular permeability increase by ultrasound-activated microbubbles in a developed microvessel-on-a-chip model aids the safe and efficient development of microbubble-mediated drug transport. ... Read more