Dependence of sonoporation efficiency on microbubble size: An in vitro monodisperse microbubble study.

Sonoporation is the process where intracellular drug delivery is facilitated by ultrasound-driven microbubble oscillations. Several mechanisms have been proposed to relate microbubble dynamics to sonoporation including shear and normal stress. The present work aims to gain insight into the role of microbubble size on sonoporation and thereby into the relevant mechanism(s) of sonoporation. To this end, we measured the sonoporation efficiency while varying microbubble size using monodisperse microbubble suspensions. Sonoporation experiments were performed in vitro on cell monolayers using a single ultrasound pulse with a fixed frequency of 1 MHz while the acoustic pressure amplitude and pulse length were varied at 250, 500, and 750 kPa, and 10, 100, and 1000 cycles, respectively. Sonoporation efficiency was quantified using flow cytometry by measuring the FITC-dextran (4 kDa and 2 MDa) fluorescence intensity in 10,000 cells per experiment to average out inherent variations in the bioresponse. Using ultra-high-speed imaging at 10 million frames per second, we demonstrate that the bubble oscillation amplitude is nearly independent of the equilibrium bubble radius at acoustic pressure amplitudes that induce sonoporation (≥ 500 kPa). However, we show that sonoporation efficiency is strongly dependent on the equilibrium bubble size and that under all explored driving conditions most efficiently induced by bubbles with a radius of 4.7 µm. Polydisperse microbubbles with a typical ultrasound contrast agent size distribution perform almost an order of magnitude lower in terms of sonoporation efficiency than the 4.7-µm bubbles. We elucidate that for our system shear stress is highly unlikely the mechanism of action. By contrast, we show that sonoporation efficiency correlates well with an estimate of the bubble-induced normal stress.

Monodisperse versus Polydisperse Ultrasound Contrast Agents: In Vivo Sensitivity and safety in Rat and Pig.

Recent advances in the field of monodisperse microbubble synthesis by flow focusing allow for the production of foam-free, highly concentrated and monodisperse lipid-coated microbubble suspensions. It has been found that in vitro, such monodisperse ultrasound contrast agents (UCAs) improve the sensitivity of contrast-enhanced ultrasound imaging. Here, we present the first in vivo study in the left ventricle of rat and pig with this new monodisperse bubble agent. We systematically characterize the acoustic sensitivity and safety of the agent at an imaging frequency of 2.5 MHz as compared with three commercial polydisperse UCAs (SonoVue/Lumason, Definity/Luminity and Optison) and one research-grade polydisperse agent with the same shell composition as the monodisperse bubbles. The monodisperse microbubbles, which had a diameter of 4.2 µm, crossed the pulmonary vasculature, and their echo signal could be measured at least as long as that of the polydisperse UCAs, indicating that microfluidically formed monodisperse microbubbles are stable in vivo. Furthermore, it was found that the sensitivity of the monodisperse agent, expressed as the mean echo power per injected bubble, was at least 10 times higher than that of the polydisperse UCAs. Finally, the safety profile of the monodisperse microbubble suspension was evaluated by injecting 400 and 2000 times the imaging dose, and neither physiologic nor pathologic changes were found, which is a first indication that monodisperse lipid-coated microbubbles formed by flow focusing are safe for in vivo use. The more uniform acoustic response and corresponding increased imaging sensitivity of the monodisperse agent may boost emerging applications of microbubbles and ultrasound such as molecular imaging and therapy.

Synthesis and characterization of transiently stable albumin-coated microbubbles via a flow-focusing microfluidic device.

We describe a method for synthesizing albumin-shelled, large-diameter (>10 µm), transiently stable microbubbles using a flow-focusing microfluidic device (FFMD). The microfluidic device enables microbubbles to be produced immediately before insonation, thus relaxing the requirements for stability. Both reconstituted fractionated bovine serum albumin (BSA) and fresh bovine blood plasma were investigated as shell stabilizers. Microbubble coalescence was inhibited by the addition of either dextrose or glycerol and propylene glycol. Microbubbles were observed to have an acoustic half-life of approximately 6 s. Microbubbles generated directly within a vessel phantom containing flowing blood produced a 6.5-dB increase in acoustic signal within the lumen. Microbubbles generated in real time upstream of in vitro rat aortic smooth muscle cells under physiologic flow conditions successfully permeabilized 58% of the cells on insonation at a peak negative pressure of 200 kPa. These results indicate that transiently stable microbubbles produced via flow-focusing microfluidic devices are capable of image enhancement and drug delivery. In addition, successful microbubble production with blood plasma suggests the potential to use blood as a stabilizing shell.

Liquid Flooded Flow-Focusing Microfluidic Device for in situ Generation of Monodisperse Microbubbles.

Current microbubble-based ultrasound contrast agents are administered intravenously resulting in large losses of contrast agent, systemic distribution, and strict requirements for microbubble longevity and diameter size. Instead we propose in situ production of microbubbles directly within the vasculature to avoid these limitations. Flow focusing microfluidic devices (FFMDs) are a promising technology for enabling in situ production as they can produce microbubbles with precisely controlled diameters in real-time. While the microfluidic chips are small, the addition of inlets and interconnects to supply the gas and liquid phase greatly increases the footprint of these devices preventing the miniaturization of FFMDs to sizes compatible with medium and small vessels. To overcome this challenge, we introduce a new method for supplying the liquid (shell) phase to an FFMD that eliminates bulky interconnects. A pressurized liquid-filled chamber is coupled to the liquid inlets of an FFMD, which we term a flooded FFMD. The microbubble diameter and production rate of flooded FFMDs were measured optically over a range of gas pressures and liquid flow rates. The smallest FFMD manufactured measured 14.5 × 2.8 × 2.3 mm. A minimum microbubble diameter of 8.1 ± 0.3 µm was achieved at a production rate of 450,000 microbubbles/s (MB/s). This represents a significant improvement with respect to any previously reported result. The flooded design also simplifies parallelization and production rates of up to 670,000 MB/s were achieved using a parallelized version of the flooded FFMD. In addition, an intravascular ultrasound (IVUS) catheter was coupled to the flooded FFMD to produce an integrated ultrasound contrast imaging device. B-mode and IVUS images of microbubbles produced from a flooded FFMD in a gelatin phantom vessel were acquired to demonstrate the potential of in situ microbubble production and real-time imaging. Microbubble production rates of 222,000 MB/s from a flooded FFMD within the vessel lumen provided a 23 dB increase in B-mode contrast. Overall, the flooded design is a critical contribution towards the long- term goal of utilizing in situ produced microbubbles for contrast enhanced ultrasound imaging of, and drug delivery to, the vasculature.