This paper presents a new manufacturing method to generate monodisperse microbubble contrast agents with polydispersity index (σ) values of <2% through microfluidic flow-focusing. Micronsized lipid shell-based perfluorocarbon (PFC) gas microbubbles for use as ultrasound contrast agents were produced using this method. The poly(dimethylsiloxane) (PDMS)-based devices feature expanding nozzle geometry with a 7 μm orifice width, and are robust enough for consistent production of microbubbles with runtimes lasting several hours. With high-speed imaging, we characterized relationships between channel geometry, liquid flow rate Q, and gas pressure P in controlling bubble sizes. By a simple optimization of the channel geometry and Q and P, bubbles with a mean diameter of <5 μm can be obtained, ideal for various ultrasonic imaging applications. This method demonstrates the potential of microfluidics as an efficient means for custom-designing ultrasound contrast agents with precise size distributions, different gas compositions and new shell materials for stabilization, and for future targeted imaging and therapeutic applications.

Encapsulated microbubble contrast agents incorporating an adhesion ligand in the microbubble shell are used for molecular imaging with ultrasound. Currently available microbubble agents are produced with techniques that result in a large size variance. Detection of these contrast agents depends on properties related to the microbubble diameter such as resonant frequency, and current ultrasound imaging systems have bandwidth limits that reduce their sensitivity to a polydisperse contrast agent population. For ultrasonic molecular imaging, in which only a limited number of targeted contrast agents may be retained at the site of pathology, it is important to optimize the sensitivity of the imaging system to the entire population of contrast agent. This article presents contrast agents with a narrow size distribution that are targeted for molecular imaging applications. The production of a functionalized, lipid-encapsulated, microbubble contrast agent with a monodisperse population is demonstrated, and we evaluate parameters that influence the size distribution and demonstrate initial acoustic testing.

The dynamic processes impacting the size distributions of lipid-encapsulated microbubbles formed by flow-focusing were observed by video optical microscopy. Parameters studied included the filling gas, gas saturating the surrounding solution, and microbubble size (initial size 2-12 μm) to simulate typical laboratory conditions. Typically, dissolution or growth, followed by Ostwald ripening at a collection cover glass, were observed and quantified. However, in the case of small nitrogen-filled microbubbles surrounded by an air-saturated solution, Ostwald ripening was avoided for at least 9 h. These bubbles had a final size distribution of 1.5 ± 0.1 μm. This work suggests that lipidencapsulated microbubbles formed by flow-focusing should be given sufficient time to reach a terminal size before coming into contact with each other. These long-lived mondisperse microbubbles should be of interest in ultrasound contrast agents, microfabrication, food, and research applications.

In this letter, the long-term stabilization of monodisperse microbubbles produced by flow focusing is demonstrated using lipid encapsulation. Fluorescence microscopy, high-speed camera imaging, and particle size analysis were used to investigate the roles of lipid phase behavior, dissolution, Ostwald ripening, and coalescence in the stability of microbubbles formed by flow focusing. It was found that these behaviors were controlled through compositional changes with respect to lipid, emulsifier, and viscosity agents. Microbubbles coated with lipid and PEG emulsifier in a viscous solution were found to contain an extremely narrow size distribution (diameter av = 51 μm, standard deviation = 4 μm), which was maintained for up to several months.

The most common type of ultrasound contrast agents are encapsulated microbubbles, typically 1 to 5 microns in diameter. These microbubbles are injected into the bloodstream to provide image enhancement during an ultrasound examination. Because of their compressibility, these microbubbles are inherently sensitive to changes in pressure. For imaging, this is beneficial in that these microbubbles oscillate in an acoustic field and allow imaging systems to detect their response uniquely from tissue. However, this sensitivity also means that microbubbles can be readily destroyed by significant hydrostatic pressure. Injection of these microbubbles through a small-gauge catheter, such as is sometimes performed in small animal imaging studies, can result in microbubble destruction. In this manuscript, the effects of microbubble injection through catheters of varying diameter are examined. Our results indicate that the concentration and size distribution of microbubbles can be substantially altered in cases of rapid injection through small-gauge needles.