Kanaka Hettiarachchi

Tailoring the Size Distribution of Ultrasound Contrast Agents: Possible Method for Improving Sensitivity in Molecular Imaging

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.

Maintaining monodispersity in a microbubble population formed by flow-focusing.

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 microm) 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 microm. This work suggests that lipid-encapsulated 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.

Controllable microfluidic synthesis of multiphase drug-carrying lipospheres for site-targeted therapy

We report the production of micrometer‐sized gas‐filled lipospheres using digital (droplet‐based) 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 PDMS‐based microfluidic devices that feature a combined dual hydrodynamic flow‐focusing region and expanding nozzle geometry with a narrow orifice. Spherical vehicles are formed through flow‐focusing 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 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.

Acoustic responses of monodisperse lipid-encapsulated microbubble contrast agents produced by flow focusing.

Lipid-encapsulated microbubbles are used as contrast agents in ultrasound imaging. Currently available commercially made contrast agents have a polydisperse size distribution. It has been hypothesised that improved imaging sensitivity could be achieved with a uniform microbubble radius. We have recently developed microfluidics technology to produce contrast agents with a nearly monodisperse distribution. In this manuscript, we analyze echo responses from individual microbubbles from monodisperse populations in order to establish the relationship between scattered echo, microbubble radius, and excitation frequency. Simulations of bubble response from a modified Rayleigh-Plesset type model corroborate experimental data. Results indicate that microbubble echo response can be greatly increased by optimal combinations of microbubble radius and acoustic excitation frequency. These results may have a significant impact in the formulation of contrast agents to improve ultrasonic sensitivity.

Formulation of Monodisperse Contrast Agents in Microfluidic Systems for Ultrasonic Imaging

We have successfully demonstrated a microfluidic flow-focusing method to produce monodisperse contrast agents with a mean diameter of 4.0 microns, which is optimal for current ultrasonic imaging techniques. Currently available microbubble contrast agents are produced with agitation techniques, and the random nature of this process results in a highly polydisperse size distribution. PDMS-based microfluidic flow-focusing systems are a cost-effective means to easily and rapidly produce bubbles with a diameter of a few microns. Our device uses expanding nozzle geometry, focusing the bubble break-off location to one single point located at the orifice, which enables the formation of monodisperse bubbles. The geometry of the channel junctions in addition to the liquid and gas flow rates are used to control the bubble sizes. Using this technique, the microbubble diameter can be easily tailored to produce contrast agents with a precise size distribution optimized for various ultrasonic imaging applications

Acoustic characterization of individual monodisperse contrast agents with an optical-acoustical system

Contrast agents that are available commercially have a polydisperse size distribution. It has been hypothesized that a uniform microbubble diameter might lead to improved imaging sensitivity. Using microfluidics technology that we have developed recently, we have been able to generate contrast agents with a nearly monodisperse size distribution. Echo responses from individual microbubbles from both monodisperse and polydisperse populations were analyzed in order to establish the relationship between scattered echo, microbubble diameter, and excitation frequency. Our experimental data were in very good agreement with simulations of bubble response from a modified Rayleigh-Plesset type model. Additionally, we performed in vivo experiments for non-targeted perfusion imaging. Simulations and experiments indicate that depending on microbubble diameter, excitation frequency, and distribution uniformity, significant differences in microbubble echo amplitude can be generated and that monodisperse contrast agents can be detected with greater amplitude than polydisperse agents under optimized conditions. These findings might have a substantial impact in the formulation of contrast agents to enhance ultrasonic sensitivity.

Ultrasonic analysis of precision-engineered acoustically active lipospheres produced by microfluidic

The development of a “magic bullet” that could carry therapeutic dose of drug to a target organ or tumor with high specificity is the ideal goal of targeted drug delivery. Acoustically active drug carriers must possess a layer with drug-carrying capacity, similar to a liposome, yet at the same time, they must have a core with significantly different density and compressibility than the surrounding media - such as a gas. Factors such as consistent response to acoustic pulses and consistent loading per particle are important characteristics for reliable delivery. Here, we utilize microfluidic technology to precision engineer acoustically-active drug delivery vehicles. Microfluidic multi-layer flow focusing enables production of acoustically active lipospheres (AALs) with nearly identical diameter. We perform ultrasonic interrogation of these multi layer vehicles as they are produced to determine their acoustic activity and diameter consistency. Acoustic response from lipospheres was measured to be on the same order of magnitude as responses from thin-wall lipid shelled contrast agents, indicating the oil layer did not produce notable damping effects on the acoustic scattering. We hypothesize that based on nearly identical echo signatures, that it will be easier to optimize ultrasound radiation-force mediated concentration and acoustically-mediated drug release to affect all AALs similarly.