Ali H. Dhanaliwala

Enhanced Intracellular Delivery of a Model Drug Using Microbubbles Produced by a Microfluidic Device

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 bloodstream 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 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. Microbubbles generated by the flow-focusing microfluidic device 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 s, 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.

Focused Ultrasound-Mediated Drug Delivery From Microbubbles Reduces Drug Dose Necessary for Therapeutic Effect on Neointima Formation—Brief Report

Objective—We hypothesized that (1) neointima formation in a rat carotid balloon injury model could be reduced in vivo following targeted ultrasound delivery of rapamycin 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 intravenously and ruptured at the site of injury with ultrasound. Compared with nontreated 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.

Real-time targeted molecular imaging using singular value spectra properties to isolate the adherent microbubble signal

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 a new 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⁻¹ 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 a 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 a 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 the 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 a larger area under the receiver operating characteristic curve (P < 0.05, n = 4).

Production rate and diameter analysis of spherical monodisperse microbubbles from two-dimensional, expanding-nozzle flow-focusing microfluidic devices

Flow-focusing microfluidic devices (FFMDs) can produce microbubbles (MBs) with precisely controlled diameters and a narrow size distribution. In this paper, poly-dimethyl-siloxane based, rectangular-nozzle, two-dimensional (2-D) planar, expanding-nozzle FFMDs were characterized using a high speed camera to determine the production rate and diameter of Tween 20 (2% v/v) stabilized MBs. The effect of gas pressure and liquid flow rate on MB production rate and diameter was analyzed in order to develop a relationship between FFMD input parameters and MB production. MB generation was observed to transition through five regimes at a constant gas pressure and increasing liquid flow rate. Each MB generation event (i.e., break-off to break-off) was further separated into two characteristic phases: bubbling and waiting. The duration of the bubbling phase was linearly related to the liquid flow rate, while the duration of the waiting phase was related to both liquid flow rate and gas pressure. The MB production rate was found to be inversely proportional to the sum of the bubbling and waiting times, while the diameter was found to be proportional to the product of the gas pressure and bubbling time.

In vivo imaging of microfluidic-produced microbubbles.

Microfluidics-based production of stable microbubbles for ultrasound contrast enhancement or drug/gene delivery allows for precise control over microbubble diameter but at the cost of a low production rate. In situ microfluidic production of microbubbles directly in the vasculature may eliminate the necessity for high microbubble production rates, long stability, or small diameters. Towards this goal, we investigated whether microfluidic-produced microbubbles directly administered into a mouse tail vein could provide sufficient ultrasound contrast. Microbubbles composed of nitrogen gas and stabilized with 3 % bovine serum albumin and 10 % dextrose were injected for 10 seconds into wild type C57BL/6 mice, via a tail-vein catheter. Short-axis images of the right and left ventricle were acquired at 12.5 MHz and image intensity over time was analyzed. Microbubbles were produced on the order of 105 microbubbles/s and were observed in both the right and left ventricles. The median rise time, duration, and decay time within the right ventricle were 2.9, 21.3, and 14.3 s, respectively. All mice survived the procedure with no observable respiratory or heart rate distress despite microbubble diameters as large as 19 μm.

Assessing and improving acoustic radiation force image quality using a 1.5-D transducer design

A 1.5-D transducer array was proposed to improve acoustic radiation force impulse (ARFI) imaging signal-to-noise ratio (SNRARFI) and image contrast relative to a conventional 1-D array. To predict performance gains from the proposed 1.5-D transducer array, an analytical model for SNRARFI upper bound was derived. The analytical model and 1.5-D ARFI array were validated using a finite element model-based numerical simulation framework. The analytical model demonstrated good agreement with numerical results (correlation coefficient = 0.995), and simulated lesion images yielded a significant (2.92 dB; p <; 0.001) improvement in contrast-to-noise ratio when rendered using the 1.5-D ARFI array.

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.

Synthesis of albumin microbubbles using a microfluidic device for real-time imaging and therapeutics

A method for producing monodisperse albumin stabilized microbubbles (MBs) using a flow-focusing microfluidic device (FFMD) is introduced. This method allows for localized delivery of short life-time microbubbles with a biocompatible shell, thereby potentially improving patient safety. In this study, microbubbles stabilized with bovine serum albumin (BSA) are characterized for microbubble coalescence, size, and production rate using a high speed camera. Microbubbles were produced with diameters between 10-20 μm and production rates up to 6×105 MB/s were achieved. Microbubble diameter was observed to decrease linearly (R2 > 0.99) with liquid flow rate. Microbubble stability was evaluated acoustically using a clinical ultrasound scanner. The image intensity of a well-mixed solution containing 13 μm microbubbles returned to baseline from peak intensity within 30s. Delivery of the membrane impermeable fluorophore calcein was demonstrated using microbubbles produced in situ within an in vitro flow chamber. 11 μm diameter microbubbles insonated at a peak negative pressure (PNP) of 200 kPA resulted in 58.0% of cells to uptake calcein. To demonstrate the ability to stabilize microbubbles directly with blood, plasma was separated from whole bovine blood, input into the FFMD, and used to produce microubbbles. These plasma microbubbles were imaged under flow in a gelatin flow phantom and demonstrated a 6.5 dB increase in lumen contrast.

Parallel output, liquid flooded flow-focusing microfluidic device for generating monodisperse microbubbles within a catheter

A new method for supplying the liquid phase to a flow focusing microfluidic device (FFMD), designed for the production of monodisperse microbubbles (MBs), is introduced. The FFMD is coupled to a pressurized liquid-filled chamber, avoiding the need for dedicated liquid phase tubing or interconnects from the external field to the microfluidics device. This method significantly reduces the complexity of FFMD fabrication and simplifies the parallelization of FFMDs - an important consideration for increasing MB production rate. Using this new method, flooded FFMDs were fabricated and MB diameter and production rate were measured. The minimum MB size produced was 7.1±0.5 μm. The maximum production rate from a single nozzle FFMD was 333,000, MB/s. Production was increased 1.5-fold using a two nozzle, parallelized device, for a maximum production rate of approximately 500,000 MB/s. In addition to increased production, the flooded design allows for miniaturization, with the smallest FFMD measuring 14.5 × 2.8 × 2.3 mm. Finally, B-mode and intravascular ultrasound images were obtained, highlighting the potential for flooded FFMDs to generate microbubbles in situ in a catheter and immediately thereafter image the same MBs in a target blood vessel.

Acoustically active red blood cell carriers for ultrasound-triggered drug delivery with photoacoustic tracking

As a biological alternative to conventional microbubble and droplet based ultrasound therapeutic agents, red blood cell (RBC) carriers have a potentially higher drug payload and extended circulation lifetime. RBC carriers, however, lack remote triggering for active release of payload. In this work we conjugate perfluoropentane (PFP) and perfluorobutane (PFB) nanodroplets (NDs) to RBC carriers and show ultrasound-triggered release of a model drug from carrier RBCs. With a fluorescence release assay, we achieve a release of 55.0 ± 5.0% and 46.5 ± 10.8% for RBC carriers with PFP or PFB NDs, respectively. We load magnetic nanoparticles for magnetic targeting and the FDA approved optically absorbing dye, indocyanine green (ICG), into RBCs (ICG-RBCs) for photoacoustic (PA) imaging. ICG-RBCs generated a 17 dB increase in PA signal over background and lysis of ICG-RBCs was confirmed by PA imaging. Our results demonstrate the acoustic release mechanism of RBC drug carriers, and the ability to monitor these agents in real-time.