Joseph P. Kilroy

Multispectral photoacoustic microscopy based on an optical-acoustic objective.

We have developed reflection-mode multispectral photoacoustic microscopy (PAM) based on a novel optical–acoustic objective that integrates a customized ultrasonic transducer and a commercial reflective microscope objective into one solid piece. This technical innovation provides zero chromatic aberration and convenient confocal alignment of the optical excitation and acoustic detection. With a wavelength-tunable optical-parametric-oscillator laser, we have demonstrated multispectral PAM over an ultrabroad spectral range of 270–1300 nm. A near-constant lateral resolution of ∼2.8 μm is achieved experimentally. Capitalizing on the consistent performance over the ultraviolet, visible, and near-infrared range, multispectral PAM enables label-free concurrent imaging of cell nucleus (DNA/RNA contrast at 270 nm), blood vessel (hemoglobin contrast at 532 nm), and sebaceous gland (lipid contrast at 1260 nm) at the same spatial scale in a living mouse ear.

Intravascular ultrasound catheter to enhance microbubble-based drug delivery via acoustic radiation force

Previous research has demonstrated that acoustic radiation force enhances intravascular microbubble adhesion to blood vessels in the presence of flow for molecular-targeted ultrasound imaging and drug delivery. A prototype acoustic radiation force intravascular ultrasound (ARFIVUS) catheter was designed and fabricated to displace a microbubble contrast agent in flow representative of conditions encountered in the human carotid artery. The prototype ARFIVUS transducer was designed to match the resonance frequency of 1.4- to 2.6-μm-diameter microbubbles modeled by an experimentally verified 1-D microbubble acoustic radiation force translation model. The transducer element was an elongated Navy Type I (hard) lead zirconate titanate (PZT) ceramic designed to operate at 3 MHz. Fabricated devices operated with center frequencies of 3.3 and 3.6 MHz with -6-dB fractional bandwidths of 55% and 50%, respectively. Microbubble translation velocities as high as 0.86 m/s were measured using a high-speed streak camera when insonating with the ARFIVUS transducer. Finally, the prototype was used to displace microbubbles in a flow phantom while imaging with a commercial 45-MHz imaging IVUS transducer. A sustained increase of 31 dB in average video intensity was measured following insonation with the ARFIVUS, indicating microbubble accumulation resulting from the application of acoustic radiation force.

Microbubble-mediated intravascular ultrasound imaging and drug delivery

Intravascular ultrasound (IVUS) provides radiation-free, real-time imaging and assessment of atherosclerotic disease in terms of anatomical, functional, and molecular composition. The primary clinical applications of IVUS imaging include assessment of luminal plaque volume and real-time image guidance for stent placement. When paired with microbubble contrast agents, IVUS technology may be extended to provide nonlinear imaging, molecular imaging, and therapeutic delivery modes. In this review, we discuss the development of emerging imaging and therapeutic applications that are enabled by the combination of IVUS imaging technology and microbubble contrast agents.

Localized in Vivo Model Drug Delivery with Intravascular Ultrasound and Microbubbles

An intravascular ultrasound (IVUS) and microbubble drug delivery system was evaluated in both ex vivo and in vivo swine vessel models. Microbubbles with the fluorophore DiI embedded in the shell as a model drug were infused into ex vivo swine arteries at a physiologic flow rate (105 mL/min) while a 5-MHz IVUS transducer applied ultrasound. Ultrasound pulse sequences consisted of acoustic radiation force pulses to displace DiI-loaded microbubbles from the vessel lumen to the wall, followed by higher-intensity delivery pulses to release DiI into the vessel wall. Insonation with both the acoustic radiation force pulse and the delivery pulse increased DiI deposition 10-fold compared with deposition with the delivery pulse alone. Localized delivery of DiI was then demonstrated in an in vivo swine model. The theoretical transducer beam width predicted the measured angular extent of delivery to within 11%. These results indicate that low-frequency IVUS catheters are a viable method for achieving localized drug delivery with microbubbles.

An IVUS transducer for microbubble therapies

There is interest in examining the potential of modified intravascular ultrasound (IVUS) catheters to facilitate dual diagnostic and therapeutic roles using ultrasound plus microbubbles for localized drug delivery to the vessel wall. The goal of this study was to design, prototype, and validate an IVUS transducer for microbubble-based drug delivery. A 1-D acoustic radiation force model and finite element analysis guided the design of a 1.5-MHz IVUS transducer. Using the IVUS transducer, biotinylated microbubbles were displaced in water and bovine whole blood to the streptavidin-coated wall of a flow phantom by a 1.5-MHz center frequency, peak negative pressure = 70 kPa pulse with varying pulse repetition frequency (PRF) while monitoring microbubble adhesion with ultrasound. A fit was applied to the RF data to extract a time constant (π). As PRF was increased in water, the time constant decreased (π = 32.6 s, 1 kHz vs. π = 8.2 s, 6 kHz), whereas in bovine whole blood an adhesion-no adhesion transition was found for PRFs ≥ 8 kHz. Finally, a fluorophore was delivered to an ex vivo swine artery using microbubbles and the IVUS transducer, resulting in a 6.6-fold increase in fluorescence. These results indicate the importance of PRF (or duty factor) for IVUS acoustic radiation force microbubble displacement and the potential for IVUS and microbubbles to provide localized drug delivery.

Ultrasound catheter for microbubble based drug delivery

An intravascular ultrasound (IVUS) catheter was designed to guide and locally deliver microbubbles carrying therapeutic agents. Optimal insonation parameters were determined by modeling microbubble translational displacements using the coupled 1D Rayleigh-Plesset and Drag-displacement equations. A transducer assembly was designed based on the results of microbubble simulations and a Finite Element Analysis (FEA) Model. The transducer components were fabricated then assembled into a 1.4 mm (OD) catheter tube. Hydrophone tests were performed to measure transducer output and the resulting output was compared to the FEA model. The final catheter was tested for gene delivery effectiveness by transfecting cells with a cytomegalovirus - plasmid kinase (CMV-PK) Red plasmid DNA. Wall-less flow phantom experiments were also performed to demonstrate the effectiveness of translating microbubbles across a vessel using ultrasound radiation force by measuring change in image intensity of a B-mode scan of the vessel. The resulting change in image intensity was approximately 15 dB. A follow-up experiment with the wall-less flow phantom was performed where microbubbles were initially translated and then burst, resulting in an intensity change of up to 12 dB and then a decrease after destruction back down to 0 dB.

Triple function IVUS: Diagnostic imaging, radiation force, and therapeutic for microbubble-based drug delivery

A prototype triple function intravascular ultrasound (IVUS) catheter, providing three operating frequencies, was designed, fabricated, and tested. The prototype was characterized by collecting one way acoustic output and beam profiles. The elongated acoustic radiation force (ARF) element has a center frequency of 3.6 MHz and a -6 dB fractional bandwidth of 38%. The therapeutic and imaging element (TIE) has a center frequency of 9 MHz and -6 dB fractional bandwidth of 47% with a width mode resonance of 1.9 MHz. The maximum recorded peak negative pressure at 1.5 MHz was 600kPa for the TIE. The displacement of microbubbles due to the ARF element was optically measured and compared to a 1-D microbubble displacement model. Average microbubble velocities of up to 0.6 m/s were measured using a single 280 kPa 20 cycle 3 MHz Gaussian ramped sinusoidal pulse. A wire target was imaged with the IVUS prototype in a wall-less flow phantom.

FEA modeling of CMUT with membrane stand-off structures to enable selectable frequency-mode operation

A selectable, dual-frequency, capacitive micro- machined ultrasonic transducer (CMUT) designed for both high-frequency imaging and low-frequency therapeutic effect is presented. A validated finite element analysis (FEA) CMUT model was used to examine the performance of the proposed dual-frequency transducer. CMUT device simulations were used to design a hybrid device incorporating stand-off structures that divide a large, low-frequency membrane into smaller, high-frequency sub-membranes when the membrane is partially collapsed so that the stand-offs contact the substrate. In low-frequency operation, simulations indicated that the peak negative pressure achieved by the hybrid device, when biased by 30.0 VDC and excited by a 2-MHz signal with 30.0 V amplitude, exceeded 190 kPa, which is sufficient for microbubble rupture. Low-frequency mode bandwidth was 93% at a center frequency of 2.1 MHz. In the high-frequency mode of operation, the device was excited by 175 Vdc and 87.5 Vac, which generated a peak negative pressure of 247 kPa. Device center frequency was 44.1 MHz with a - 6-dB fractional bandwidth of 42%.

Large diameter microbubbles produced by a catheter-sized microfluidic device for sonothrombolysis applications

Therapeutic approaches that enhance thrombolysis by combining tissue plasminogen activator (tPA), ultrasound (US), and/or microbubbles (MBs) are known generally as sonothrombolysis techniques. To date, sonothrombolysis clinical trials and experimental investigations have primarily utilized commercially available MB formulations (or derivatives thereof) with MB diameters generally in the range 1 - 4 μm. The restriction on MB diameter is due to a risk of gas emboli formation, which has left MBs outside of this diameter range virtually unexplored for sonothrombolysis applications. However, it is broadly understood that large MBs confer larger bioeffects when excited acoustically, as has been shown in sonoporation, blood brain barrier disruption, and sonothrombolysis applications. In support of the hypothesis that large MBs confer enhanced therapeutic effects, we demonstrate that MBs with diameters between 10 - 20 μm achieve a 4.5-fold increase in in vitro sonothrombolysis rates compared to MBs with diameters between 1 - 4 μm. In addition, we present the development of a catheter (1.5 mm diameter) containing a flow-focusing microfluidic device (FFMD) capable of producing large-diameter MBs suitable for catheter-directed sonothrombolysis applications. The microfluidically-produced MBs are comprised of N2 gas and a weak albumin/dextrose shell, which confers short MB half-lives and reduces the risk of gas emboli formation. Finally, we present the results of administering microfluidically produced MBs directly into the rat brain to demonstrate that large MBs with short lifetimes are safe for in vivo deployment.

Multifunction intravascular ultrasound for microbubble based drug delivery

A compact, single element, mechanically rotated intravascular ultrasound (IVUS) transducer for microbubble-based drug delivery and imaging has been designed and fabricated. This device provides displacement, delivery, and imaging capabilities to enable microbubble-based delivery of a therapeutic drug and delivery verification. The IVUS transducer was designed and fabricated to operate at a center frequency of 7 MHz. Acoustic radiation radiation force displacement of microbubbles, fluorophore delivery using microbubbles in vitro and imaging capabilities were demonstrated. Finally, a fluorescent model drug was delivered under physiological flow conditions to an ex vivo porcine artery using the prototype IVUS.