Ultrasound-based real-time molecular imaging in large blood vessels holds promise for early detection and diagnosis of various important and significant diseases, such as stroke, atherosclerosis, and cancer. Central to the success of this imaging technique is the isolation of ligand-receptor bound adherent microbubbles from free microbubbles and tissue structures. In this paper, we present an alternative approach, termed singular spectrum-based targeted molecular (SiSTM) imaging, which separates signal components using singular value spectra content over local regions of complex echo data. Simulations were performed to illustrate the effects of acoustic target motion and harmonic energy on SiSTM imaging-derived measurements of statistical dimensionality. In vitro flow phantom experiments were performed under physiologically realistic conditions (2.7 cm/s flow velocity and 4 mm diameter) with targeted and non-targeted phantom channels. Both simulation and experimental results demonstrated that the relative motion and harmonic characteristics of adherent microbubbles (i.e. low motion and large harmonics) yields echo data with dimensionality that is distinct from free microbubbles (i.e. large motion and large harmonics) and tissue (i.e. low motion and low harmonics). Experimental SiSTM images produced the expected trend of greater adherent microbubble signal in targeted versus non-targeted microbubble experiments (P < 0.05, n = 4). The location of adherent microbubbles was qualitatively confirmed via optical imaging of fluorescent DiI signal along the phantom channel walls after SiSTM imaging. In comparison with two frequency-based real-time molecular imaging strategies, SiSTM imaging provided significantly higher image contrast (P < 0.001, n = 4) and larger area under the receiver operating characteristic curve (P < 0.05, n = 4).

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.

Focal drug delivery to a vessel wall facilitated by intravascular ultrasound and microbubbles holds promise as a potential therapy for atherosclerosis. Conventional methods of microbubble administration result in rapid clearance from the blood stream and significant drug loss. To address these limitations, we evaluated whether drug delivery could be achieved with transiently stable microbubbles produced in real-time and in close proximity to the therapeutic site. Rat aortic smooth muscle cells were placed in a flow chamber designed to simulate physiological flow conditions. A flow-focusing microfluidic device (FFMD) produced 8 μm diameter monodisperse microbubbles within the flow chamber, and ultrasound was applied to enhance uptake of a surrogate drug (calcein). Acoustic pressures up to 300 kPa and flow rates up to 18 mL/s were investigated. FFMD generated microbubbles were stabilized with a polyethylene glycol-40 stearate shell and had either a perfluorobutane (PFB) or nitrogen gas core. The gas core composition affected stability, with PFB and nitrogen microbubbles exhibiting half-lives of 40.7 and 18.2 seconds, respectively. Calcein uptake was observed at lower acoustic pressures with nitrogen microbubbles (100 kPa) than with PFB microbubbles (200 kPa) (p < 0.05, n ≥ 3). In addition, delivery was observed at all flow rates, with maximal delivery (> 70% of cells) occurring at a flow rate of 9 mL/s. These results demonstrate the potential of transiently stable microbubbles produced in real-time and in close proximity to the intended therapeutic site for enhancing localized drug delivery.

We present a method of synthesizing albumin-shelled, large diameter (>10 μm), transiently-stable microbubbles using a flow-focusing microfluidic device (FFMD). Microfluidic device production enables microbubbles to be produced immediately prior to 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 physiological flow conditions successfully permeabilized 58 % of the cells upon insonation at a peak negative pressure of 200 kPa. These results demonstrate 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 utilize blood as a stabilizing shell.

Objective We hypothesized that 1) neointimal formation in a rat carotid balloon injury model could be reduced in vivo following targeted ultrasound delivery of rapamycin-loaded microbubbles (RMBs), and 2) the addition of dual mode ultrasound decreases the total amount of drug needed to reduce neointima formation. Methods and Results Balloon injury was performed in rat carotids to induce neointima formation. High or low doses of RMBs were injected I.V. and ruptured at the site of injury with ultrasound. Compared to non-treated injured arteries, neointima formation was reduced by 0% and 35.9% with 108 RMBs, and by 28.7% and 34.9% in arteries treated with 109 RMBs with and without ultrasound respectively. Conclusion Without ultrasound, 10-fold higher concentrations of RMBs were needed to reduce neointima formation by at least 28%, whereas 108 RMBs combined with ultrasound were sufficient to achieve the same therapeutic effect demonstrating that this technology may have promise for localized potent drug therapy.