Johnny L. Chen

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.

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.

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.

Photoacoustic imaging of stimuli-responsive red blood cell drug delivery agents

Long circulation lifetime, large therapeutic payload, and inherent biocompatibility make engineered red blood cells (RBC) an attractive therapeutic delivery agent. However, drug release from conventional RBC carriers is diffusion limited and cannot be spatiotemporally controlled, thereby precluding targeted delivery to specific tissues. We have developed modified RBC carriers intended to function as image-guided therapeutic delivery agents. These new RBC carriers contain acoustically activatable perfluorocarbon droplets to enable ultrasound-mediated drug release, are loaded with iron oxide nanoparticles to permit magnetic targeting, and contain indocyanine green (ICG) dye to allow photoacoustic (PA) tracking of RBC accumulation. In this work, we evaluate the feasibility of detecting ICG-loaded RBCs against a whole blood background via spectroscopic PA imaging. We also characterize the increase in PA signal resultant from magnetic targeting of ICG-loaded RBCs.

In vivo acoustic contrast enhancement via simultaneous production and injection of microfluidic-produced microbubbles

Microbubble production by microfluidic devices for ultrasound contrast enhancement allows for precise control over microbubble diameter but at the cost of low production rate and poor microbubble stability. In this work, we investigated whether microbubbles produced by a microfluidic device could provide sufficient ultrasound contrast enhancement when directly injected into the mouse tail vein. Microfluidic-produced microbubbles composed of nitrogen gas and stabilized with 10% dextrose and 3% bovine serum albumin were injected for 10 seconds into the tail vein of wild type C57BL/6 mice. Short-axis ultrasound images of the right and left ventricle were acquired at 12.5 MHz and image intensity over time was analyzed. Microbubble production rates ranged between 2.5×105 and 8.3×105 microbubbles/s, and microbubble diameters were between 9.1 and 19 μm. In all cases, microbubbles were observed in both the right and left ventricle, although the average contrast enhancement was approximately 13.5 dB lower in the left ventricle than in the right ventricle. All mice survived the procedure with no observable respiratory or heart rate distress. The results of this work suggest that on-site production and immediate administration to the murine vasculature may eliminate the necessity for high microbubble production rates, long-term stability, or small microbubble diameters.

Model drug delivery by transiently stable microbubbles produced by a microfluidic device

Ultrasound-mediated drug delivery using intravascular ultrasound (IVUS) and microbubbles holds promise as a potential therapy for diseases of the vasculature. In this work, we evaluate the therapeutic potential of an on-demand microbubble production framework using a flow-focusing microfluidic device to generate microbubbles at the tip of the IVUS catheter for direct administration into the bloodstream. Rat aortic smooth muscle cells were placed in a flow chamber, 8 μm diameter monodisperse microbubbles produced within the flow chamber by a flow-focusing microfluidic device, and ultrasound was applied to enhance uptake of a surrogate drug (calcein). Flow rates up to 18 mL/s and acoustic peak-negative-pressures up to 300 kPa were investigated. Microbubbles produced by the microfluidic device were stabilized with a polyethylene glycol-40-stearate shell and had either a perfluorbutane (PFB) or nitrogen gas core. Calcein delivery was observed at lower acoustic pressures with nitrogen microbubbles than with PFB microbubbles, and maximal delivery occurred at a flow rate of 9 mL/s. These results demonstrate the potential of transiently stable microbubbles produced by a microfluidic device for enhancing localized drug delivery.