Jameel A. Feshitan

Microbubble size isolation by differential centrifugation

Microbubbles used as contrast agents for ultrasound imaging, vectors for targeted drug delivery and vehicles for metabolic gas transport require better size control for improved performance. Mechanical agitation is the only method currently available to produce microbubbles in sufficient yields for biomedical applications, but the emulsions tend to be polydisperse. Herein, we describe a study to generate lipid-coated, perfluorobutane-filled microbubbles and isolate their size fractions based on migration in a centrifugal field. Polydispersity of the freshly sonicated suspension was characterized by particle sizing and counting through light obscuration/scattering and electrical impedance sensing, fluorescence and bright-field microscopy and flow cytometry. We found that the size distribution was multimodal. Smaller microbubbles were more abundant. Differential centrifugation was used to successfully isolate the 1-2 and 4-5 mum diameter fractions. Isolated microbubbles were stable over two days. After two weeks, however, more dilute suspensions (<1 vol%) were susceptible to Ostwald ripening. For example, 4-5 mum microbubbles disintegrated into 1-2 mum microbubbles. This latter observation indicated the existence of an optimally stable diameter in the 1-2 mum range for these lipid-coated microbubbles. Overall, differential centrifugation provided a rapid and robust means for size selection and reduced polydispersity of lipid-coated microbubbles.

Microbubble-Size Dependence of Focused Ultrasound-Induced Blood–Brain Barrier Opening in Mice In Vivo

The therapeutic efficacy of neurological agents is severely limited, because large compounds do not cross the blood-brain barrier (BBB). Focused ultrasound (FUS) sonication in the presence of microbubbles has been shown to temporarily open the BBB, allowing systemically administered agents into the brain. Until now, polydispersed microbubbles (1-10 ¿m in diameter) were used, and, therefore, the bubble sizes better suited for inducing the opening remain unknown. Here, the FUS-induced BBB opening dependence on microbubble size is investigated. Bubbles at 1-2 and 4-5 ¿m in diameter were separately size-isolated using differential centrifugation before being systemically administered in mice (n = 28). The BBB opening pressure threshold was identified by varying the peak-rarefactional pressure amplitude. BBB opening was determined by fluorescence enhancement due to systemically administered, fluorescent-tagged, 3-kDa dextran. The identified threshold fell between 0.30 and 0.46 MPa in the case of 1-2 ¿m bubbles and between 0.15 and 0.30 MPa in the 4-5 ¿m case. At every pressure studied, the fluorescence was greater with the 4-5 ¿m than with the 1-2 ¿m bubbles. At 0.61 MPa, in the 1-2 ¿m bubble case, the fluorescence amount and area were greater in the thalamus than in the hippocampus. In conclusion, it was determined that the FUS-induced BBB opening was dependent on both the size distribution in the injected microbubble volume and the brain region targeted.

Effect of microbubble size on fundamental mode high frequency ultrasound imaging in mice.

High-frequency ultrasound imaging using microbubble (MB) contrast agents is becoming increasingly popular in pre-clinical and small animal studies of anatomy, flow and vascular expression of molecular epitopes. Currently, in vivo imaging studies rely on highly polydisperse microbubble suspensions, which may provide a complex and varied acoustic response. To study the effect of individual microbubble size populations, microbubbles of 1-2 microm, 4-5 microm and 6-8 microm diameter were isolated using the technique of differential centrifugation. Size-selected microbubbles were imaged in the mouse kidney over a range of concentrations using a Visualsonics Vevo 770 ultrasound imaging system (Visualsonics, Toronto, Ontario, Canada) with a 40-MHz probe in fundamental mode. Results demonstrate that contrast enhancement and circulation persistence are strongly dependent on microbubble size and concentration. Large microbubbles (4-5 and 6-8 microm) strongly enhanced the ultrasound image with positive contrast, while 1-2 microm microbubbles showed little enhancement. For example, the total integrated contrast enhancement, measured by the area under the time-intensity curve (AUC), increased 16-fold for 6-8 microm diameter microbubbles at 5 x 10(7) MB/bolus compared with 4-5 microm microbubbles at the same concentration. Interestingly, 1-2 microm diameter microbubbles, at any concentration, did not measurably enhance the integrated ultrasound signal at tissue depth, but did noticeably attenuate the signal, indicating that they had a low scattering-to-attenuation ratio. When concentration matched, larger microbubbles were more persistent in circulation. However, when volume matched, all microbubble sizes had a similar circulation half-life. These results indicated that dissolution of the gas core plays a larger role in contrast elimination than filtering by the lungs and spleen. The results of this study show that microbubbles can be tailored for optimal contrast enhancement in fundamental mode imaging.

A microcomposite hydrogel for repeated on-demand ultrasound-triggered drug delivery

Here we develop an injectable composite system based for repeated ultrasound-triggered on-demand drug delivery. An in situ-cross-linking hydrogel maintains model drug (dye)-containing liposomes in close proximity to gas-filled microbubbles that serve to enhance release events induced by ultrasound application. Dye release is tunable by varying the proportions of the liposomal and microbubble components, as well as the duration and intensity of the ultrasound pulses in vitro. Dye is minimal at baseline. The composite shows minimal cytotoxicity in vitro, and benign tissue reaction after subcutaneous injection in rats. Ultrasound application also triggers drug release for two weeks after injection in vivo.

The mechanism of interaction between focused ultrasound and microbubbles in blood-brain barrier opening in mice

The activation of bubbles by an acoustic field has been shown to temporarily open the blood-brain barrier (BBB), but the trigger cause responsible for the physiological effects involved in the process of BBB opening remains unknown. Here, the trigger cause (i.e., physical mechanism) of the focused ultrasound-induced BBB opening with monodispersed microbubbles is identified. Sixty-seven mice were injected intravenously with bubbles of 1-2, 4-5, or 6-8 μm in diameter and the concentration of 10(7) numbers/ml. The right hippocampus of each mouse was then sonicated using focused ultrasound (1.5 MHz frequency, 100 cycles pulse length, 10 Hz pulse repetition frequency, 1 min duration). Peak-rarefactional pressures of 0.15, 0.30, 0.45, or 0.60 MPa were applied to identify the threshold of BBB opening and inertial cavitation (IC). Our results suggest that the BBB opens with nonlinear bubble oscillation when the bubble diameter is similar to the capillary diameter and with inertial cavitation when it is not. The bubble may thus have to be in contact with the capillary wall to induce BBB opening without IC. BBB opening was shown capable of being induced safely with nonlinear bubble oscillation at the pressure threshold and its volume was highly dependent on both the acoustic pressure and bubble diameter.

Theranostic Gd(III)-lipid microbubbles for MRI-guided focused ultrasound surgery

We have synthesized a biomaterial consisting of Gd(III) ions chelated to lipid-coated, size-selected microbubbles for utility in both magnetic resonance and ultrasound imaging. The macrocyclic ligand DOTA-NHS was bound to PE headgroups on the lipid shell of pre-synthesized microbubbles. Gd(III) was then chelated to DOTA on the microbubble shell. The reaction temperature was optimized to increase the rate of Gd(III) chelation while maintaining microbubble stability. ICP-OES analysis of the microbubbles determined a surface density of 7.5 × 10(5) ± 3.0 × 10(5) Gd(III)/μm(2) after chelation at 50 °C. The Gd(III)-bound microbubbles were found to be echogenic in vivo during high-frequency ultrasound imaging of the mouse kidney. The Gd(III)-bound microbubbles also were characterized by magnetic resonance imaging (MRI) at 9.4 T by a spin-echo technique and, surprisingly, both the longitudinal and transverse proton relaxation rates were found to be roughly equal to that of no-Gd(III) control microbubbles and saline. However, the relaxation rates increased significantly, and in a dose-dependent manner, after sonication was used to fragment the Gd(III)-bound microbubbles into non-gas-containing lipid bilayer remnants. The longitudinal (r(1)) and transverse (r(2)) molar relaxivities were 4.0 ± 0.4 and 120 ± 18 mM(-1)s(-1), respectively, based on Gd(III) content. The Gd(III)-bound microbubbles may find application in the measurement of cavitation events during MRI-guided focused ultrasound therapy and to track the biodistribution of shell remnants.

Evaluation of Peritoneal Microbubble Oxygenation Therapy in a Rabbit Model of Hypoxemia

Alternative extrapulmonary oxygenation technologies are needed to treat patients suffering from severe hypoxemia refractory to mechanical ventilation. We previously demonstrated that peritoneal microbubble oxygenation (PMO), in which phospholipid-coated oxygen microbubbles (OMBs) are delivered into the peritoneal cavity, can successfully oxygenate rats suffering from a right pneumothorax. This study addressed the need to scale up the procedure to a larger animal with a splanchnic cardiac output similar to humans. Our results show that PMO therapy can double the survival time of rabbits experiencing complete tracheal occlusion from 6.6 ± 0.6 min for the saline controls to 12.2 ± 3.0 min for the bolus PMO-treated cohort. Additionally, we designed and tested a new peritoneal delivery system to circulate OMBs through the peritoneal cavity. Circulation achieved a similar survival benefit to bolus delivery under these conditions. Overall, these results support the feasibility of the PMO technology to provide extrapulmonary ventilation for rescue of severely hypoxic patients.

Ultrasound-modulated fluorescence based on fluorescent microbubbles

Abstract. Ultrasound-modulated fluorescence (UMF) imaging has been proposed to provide fluorescent contrast while maintaining ultrasound resolution in an optical-scattering medium (such as biological tissue). The major challenge is to extract the weakly modulated fluorescent signal from a bright and unmodulated background. UMF was experimentally demonstrated based on fluorophore-labeled microbubble contrast agents. These contrast agents were produced by conjugating N-hydroxysuccinimide (NHS)-ester-attached fluorophores on the surface of amine-functionalized microbubbles. The fluorophore surface concentration was controlled so that a significant self-quenching effect occurred when no ultrasound was applied. The intensity of the fluorescent emission was modulated when microbubbles were oscillated by ultrasound pulses, presented as UMF signal. Our results demonstrated that the UMF signals were highly dependent on the microbubbles’ oscillation amplitude and the initial surface fluorophore-quenching status. A maximum of ∼42% UMF modulation depth was achieved with a single microbubble under an ultrasound peak-to-peak pressure of 675 kPa. Further, UMF was detected from a 500-μm tube filled with contrast agents in water and scattering media with ultrasound resolution. These results indicate that ultrasound-modulated fluorescent microbubble contrast agents can potentially be used for fluorescence-based molecular imaging with ultrasound resolution in the future.

Magnetic Resonance Properties of Gd(III)-Bound Lipid-Coated Microbubbles and their Cavitation Fragments

Gas-filled microbubbles are potentially useful theranostic agents for magnetic resonance imaging-guided focused ultrasound surgery (MRIgFUS). Previously, MRI at 9.4 T was used to measure the contrast properties of lipid-coated microbubbles with gadolinium (Gd(III)) bound to lipid headgroups, which revealed that the longitudinal molar relaxivity (r(1)) increased after microbubble fragmentation. This behavior was attributed to an increase in water proton exchange with the Gd(III)-bound lipid fragments caused by an increase in the lipid headgroup area that accompanied the lipid shell monolayer-to-bilayer transition. In this article, we explore this mechanism by comparing the changes in r(1) and its transverse counterpart, r(2)*, after the fragmentation of microbubbles consisting of Gd(III) bound to two different locations on the lipid monolayer shell: the phosphatidylethanolamine (PE) lipid headgroup region or the distal region of the poly(ethylene glycol) (PEG) brush. Nuclear magnetic resonance (NMR) at 1.5 T was used to measure the contrast properties of the various microbubble constructs because this is the most common field strength used in clinical MRI. Results for the lipid-headgroup-labeled Gd(III) microbubbles revealed that r(1) increased after microbubble fragmentation, whereas r(2)* was unchanged. An analysis of PEG-labeled Gd(III) microbubbles revealed that both r(1) and r(2)* decreased after microbubble fragmentation. Further analysis revealed that the microbubble gas core enhanced the transverse MR signal (T(2)*) in a concentration-dependent manner but minimally affected the longitudinal (T(1)) signal. These results illustrate a new method for the use of NMR to measure the biomembrane packing structure and suggest that two mechanisms, proton-exchange enhancement by lipid membrane relaxation and magnetic field inhomogeneity imposed by the gas/liquid interface, may be used to detect and differentiate Gd(III)-labeled microbubbles and their cavitation fragments with MRI.

Identifying the inertial cavitation threshold in a vessel phantom using focused ultrasound and microbubbles.

Unveiling the mechanism behind the blood brain barrier using focused ultrasound (FUS) and microbubbles is essential for brain molecular delivery. Here, B‐mode imaging and rf signals were acquired to pinpoint the threshold for inertial cavitation during FUS with microbubbles. A cylindrical hole of 800 μm in diameter was generated inside a polyacrylamide gel to simulate a brain arterial vessel. Definity® (Lantheus Medical Imaging, USA) microbubbles with a 1.1–3.3 μm‐diameter and (1,2‐distearoyl‐sn‐glycero‐3‐phosphocholine) (DSPC) shelled microbubbles with a 1–2‐μm‐diameter were injected prior to sonication (frequency: 1.525 MHz; pulse length: 100 cycles; PRF: 1 kHz; pulse duration: 40 ms). The cavitation response was passively detected using a 7.5‐MHz single‐element transducer, confocal with the FUS transducer, and a one‐dimensional linear array transducer placed perpendicular to the FUS beam. The broadband spectral response of the acquired rf signals and the B‐mode images detected the occurrence and locati...