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.

Cells have been encapsulated inside lipid vesicles by using a new microfluidic lipid vesicle formulation technique. Lipid vesicles are formulated within minutes without using toxic lipid solvents. The encapsulation efficiency inside the vesicles is controlled by the microfluidic flows. Green fluorescent proteins (GFP), carcinoma cells, and bead encapsulated vesicles have mean diameters of 27.2 mum, 62.4 mum, and 55.9 mum, respectively. The variations of vesicle sizes are approximately 20% for the GFP and cell encapsulated vesicles and approximately 10% for the bead encapsulated vesicles.

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.

We report the production of micrometer-sized gas-filled lipospheres using digital microfluidics technology for chemotherapeutic drug delivery. Advantages of on-chip synthesis include a monodisperse size distribution (polydispersity index (σ) values of <5%) with consistent stability and uniform drug loading. Photolithography techniques are applied to fabricate novel PDMSbased microfluidic devices that feature a combined dual hydrodynamic flow-focusing region and expanding nozzle geometry with a narrow orifice. Spherical vehicles are formed through flowfocusing by the self-assembly of phospholipids to a lipid layer around the gas core, followed by a shear-induced break off at the orifice. The encapsulation of an extra oil layer between the outer lipid shell and inner bubble gaseous core allows the safe transport of highly hydrophobic and toxic drugs at high concentrations. Doxorubicin (Dox) entrapment is estimated at 15 mg mL −1 of particles packed in a single ordered layer. In addition, the attachment of targeting ligands to the lipid shell allows for direct vehicle binding to cancer cells. Preliminary acoustic studies of these monodisperse gas lipospheres reveal a highly uniform echo correlation of greater than 95%. The potential exists for localized drug concentration and release with ultrasound energy.