Steven Feingold

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.

Validation of Dynamic Contrast Enhanced Ultrasound in Rodent Kidneys as an Absolute Quantitative Method for Measuring Blood Perfusion

Contrast-enhanced ultrasound (CEUS) has demonstrated utility in the monitoring of blood flow in tissues, organs and tumors. However, current CEUS methods typically provide only relative image-derived measurements, rather than quantitative values of blood flow in milliliters/minute per gram of tissue. In this study, CEUS derived parameters of blood flow are compared with absolute measurements of blood flow in rodent kidneys. Additionally, the effects of contrast agent infusion rate and transducer orientation on image-derived perfusion measurements are assessed. Both wash-in curve and time-to-refill algorithms are examined. Data illustrate that for all conditions, image-derived flow measurements were well-correlated with transit-time flow probe measurements (R > 0.9). However, we report differences in the sensitivity to flow across different transducer orientations as well as the contrast analysis algorithm utilized. Results also indicate that there exists a range of contrast agent flow rates for which image-derived estimates are consistent.

An in vivo validation of the application of acoustic radiation force to enhance the diagnostic utility of molecular imaging using 3-d ultrasound.

For more than a decade, the application of acoustic radiation force (ARF) has been proposed as a mechanism to increase ultrasonic molecular imaging (MI) sensitivity in vivo. Presented herein is the first noninvasive in vivo validation of ARF-enhanced MI with an unmodified clinical system. First, an in vitro optical-acoustical setup was used to optimize system parameters and ensure sufficient microbubble translation when exposed to ARF. 3-D ARF-enhanced MI was then performed on 7 rat fibrosarcoma tumors using microbubbles targeted to α(v)β₃ and nontargeted microbubbles. Low-amplitude (<25 kPa) 3-D ARF pulse sequences were tested and compared with passive targeting studies in the same animal. Our results demonstrate that a 78% increase in image intensity from targeted microbubbles can be achieved when using ARF relative to the passive targeting studies. Furthermore, ARF did not significantly increase image contrast when applied to nontargeted agents, suggesting that ARF did not increase nonspecific adhesion.

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.

Improving the quantitative ability of contrast enhanced ultrasound perfusion imaging: Effect of contrast administration rate and imaging plane orientation

Contrast-enhanced ultrasound (CEUS) has demonstrated utility in the monitoring of blood flow in tissues, organs, and tumors. Current CEUS methods typically provide only relative image-derived measurements, rather than quantitative values of blood flow in milliliters/minute per gram of tissue. The purpose of this study is to validate CEUS-derived measurements of renal blood flow (RBF) with absolute values of ml/min per gram of tissue based on a calibrated flow probe. New information about the effects of parameters such as contrast agent administration rate and transducer positioning on two different image-derived measurements are provided.

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.

Three dimensional perfusion imaging in the rat kidney using ultrasound contrast

Recently, there has been great interest in the use of ultrasound contrast to generate quantitative information about tissue perfusion. Using low MI pulse sequences and a clearance — refill approach, it is possible to create quantitative time-to-refill maps of tissue correlating to blood perfusion rate. One limitation of 2D perfusion imaging is that only information from a single slice of tissue is gathered. Vascular inhomogeneity, common in many tumor types, makes perfusion estimates inconsistent unless the same region is imaged repeatedly. Our objective was to evaluate errors in 2D quantitative in-vivo perfusion estimates due to differences in transducer positioning compared to 3D acquisitions. We also examined the effect of contrast dose on perfusion estimates. Destruction-reperfusion imaging was performed with parametric mapping of refill times and image alignment to correct for tissue motion. Images were acquired in rat kidneys using a Siemens Sequoia 512. 3D images were generated by mounting the transducer to a computer controlled linear motion stage. Changes in perfusion times were examined as a function of contrast dose, imaging plane, and in response to the vasoactive drug dopamine. Our results showed that we could differentiate perfusion changes before and after administration of dopamine, and that 3D images were more consistent than 2D acquisitions. We also observed bias caused by contrast agent infusion rate using our time-to-refill algorithm; however, in the range of 2.7 × 108 and 3.9 × 108 bubbles/min the results obtained were consistent to within 3%. 3D perfusion imaging demonstrated a significant reduction in error caused by transducer positioning and improves the reliability of quantitative perfusion time estimates in the rat kidney model.

Efficacy of perfluorobutane as a phase-change contrast agent for low-energy ultrasonic imaging

Most gas-filled ultrasound contrast agents are produced range of several microns in diameter, which limits them to flow within intravascular space. One mechanism proposed to produce extravascular imaging agents is acoustic droplet vaporization. Liquid perfluorocarbon droplets can be manufactured in the sub-micron range and can then passively diffuse through leaky tumor vasculature. It is hypothesized that once extravasated, these droplets could be converted to microbubbles in the micron range through additional energy input in the form of ultrasound, resulting in enhanced imaging contrast. Recent studies show current formulations of phase-change contrast agents in the sub-micron range may require substantial acoustic energy to vaporize, which increases the chance of bioeffects. Thus, phase-change contrast agents with reduced acoustic activation energies would have significant advantages. In this study, the generation and activation of novel phase-change contrast agents formulated with perfluorobutane is demonstrated. Perfluorobutane — normally a gas at room temperature — can be incorporated into metastable liquid sub-micron droplets with lipid encapsulation methods. The resulting droplets are shown to be acoustically vaporizable with substantially less energy than other compounds proposed for phase-change contrast agents such as perfluoropentane and perfluorohexane.