Mark A. Borden

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.

Lipid-shelled vehicles: engineering for ultrasound molecular imaging and drug delivery.

Ultrasound pressure waves can map the location of lipid-stabilized gas micro-bubbles after their intravenous administration in the body, facilitating an estimate of vascular density and microvascular flow rate. Microbubbles are currently approved by the Food and Drug Administration as ultrasound contrast agents for visualizing opacification of the left ventricle in echocardiography. However, the interaction of ultrasound waves with intravenously-injected lipid-shelled particles, including both liposomes and microbubbles, is a far richer field. Particles can be designed for molecular imaging and loaded with drugs or genes; the mechanical and thermal properties of ultrasound can then effect localized drug release. In this Account, we provide an overview of the engineering of lipid-shelled microbubbles (typical diameter 1000-10 000 nm) and liposomes (typical diameter 65-120 nm) for ultrasound-based applications in molecular imaging and drug delivery. The chemistries of the shell and core can be optimized to enhance stability, circulation persistence, drug loading and release, targeting to and fusion with the cell membrane, and therapeutic biological effects. To assess the biodistribution and pharmacokinetics of these particles, we incorporated positron emission tomography (PET) radioisotopes on the shell. The radionuclide (18)F (half-life approximately 2 h) was covalently coupled to a dipalmitoyl lipid, followed by integration of the labeled lipid into the shell, facilitating short-term analysis of particle pharmacokinetics and metabolism of the lipid molecule. Alternately, labeling a formed particle with (64)Cu (half-life 12.7 h), after prior covalent incorporation of a copper-chelating moiety onto the lipid shell, permits pharmacokinetic study of particles over several days. Stability and persistence in circulation of both liposomes and microbubbles are enhanced by long acyl chains and a poly(ethylene glycol) coating. Vascular targeting has been demonstrated with both nano- and microdiameter particles. Targeting affinity of the microbubble can be modulated by burying the ligand within a polymer brush layer; the application of ultrasound then reveals the ligand, enabling specific targeting of only the insonified region. Microbubbles and liposomes require different strategies for both drug loading and release. Microbubble loading is inhibited by the gas core and enhanced by layer-by-layer construction or conjugation of drug-entrapped particles to the surface. Liposome loading is typically internal and is enhanced by drug-specific loading techniques. Drug release from a microbubble results from the oscillation of the gas core diameter produced by the sound wave, whereas that from a liposome is enhanced by heat produced from the local absorption of acoustic energy within the tissue microenvironment. Biological effects induced by ultrasound, such as changes in cell membrane and vascular permeability, can enhance drug delivery. In particular, as microbubbles oscillate near a vessel wall, shock waves or liquid jets enhance drug transport. Mild heating induced by ultrasound, either before or after injection of the drug, facilitates the transport of liposomes from blood vessels to the tissue interstitium, thus increasing drug accumulation in the target region. Lipid-shelled vehicles offer many opportunities for chemists and engineers; ultrasound-based applications beyond the few currently in common use will undoubtedly soon multiply as molecular construction techniques are further refined.

Microbubble Compositions, Properties and Biomedical Applications

Over the last decade, there has been significant progress towards the development of microbubbles as theranostics for a wide variety of biomedical applications. The unique ability of microbubbles to respond to ultrasound makes them useful agents for contrast ultrasound imaging, molecular imaging, and targeted drug and gene delivery. The general composition of a microbubble is a gas core stabilized by a shell comprised of proteins, lipids or polymers. Each type of microbubble has its own unique advantages and can be tailored for specialized functions. In this review, different microbubbles compositions and physiochemical properties are discussed in the context of current progress towards developing novel constructs for biomedical applications, with specific emphasis on molecular imaging and targeted drug/gene delivery.

Influence of lipid shell physicochemical properties on ultrasound-induced microbubble destruction

We present the first study of the effects of monolayer shell physicochemical properties on the destruction of lipid-coated microbubbles during insonification with single, one-cycle pulses at 2.25 MHz and low-duty cycles. Shell cohesiveness was changed by varying phospholipid and emulsifier composition, and shell microstructure was controlled by postproduction processing. Individual microbubbles with initial resting diameters between 1 and 10 /spl mu/m were isolated and recorded during pulsing with brightfield and fluorescence video microscopy. Microbubble destruction occurred through two modes: acoustic dissolution at 400 and 600 kPa and fragmentation at 800 kPa peak negative pressure. Lipid composition significantly impacted the acoustic dissolution rate, fragmentation propensity, and mechanism of excess lipid shedding. Less cohesive shells resulted in micron-scale or smaller particles of excess lipid material that shed either spontaneously or on the next pulse. Conversely, more cohesive shells resulted in the buildup of shell-associated lipid strands and globular aggregates of several microns in size; the latter showed a significant increase in total shell surface area and lability. Lipid-coated microbubbles were observed to reach a stable size over many pulses at intermediate acoustic pressures. Observations of shell microstructure between pulses allowed interpretation of the state of the shell during oscillation. We briefly discuss the implications of these results for therapeutic and diagnostic applications involving lipid-coated microbubbles as ultrasound contrast agents and drug/gene delivery vehicles.

Radiation-Force Assisted Targeting Facilitates Ultrasonic Molecular Imaging

Ultrasonic molecular imaging employs contrast agents, such as microbubbles, nanoparticles, or liposomes, coated with ligands specific for receptors expressed on cells at sites of angiogenesis, inflammation, or thrombus. Concentration of these highly echogenic contrast agents at a target site enhances the ultrasound signal received from that site, promoting ultrasonic detection and analysis of disease states. In this article, we show that acoustic radiation force can be used to displace targeted contrast agents to a vessel wall, greatly increasing the number of agents binding to available surface receptors. We provide a theoretical evaluation of the magnitude of acoustic radiation force and show that it is possible to displace micron-sized agents physiologically relevant distances. Following this, we show in a series of experiments that acoustic radiation force can enhance the binding of targeted agents: The number of biotinylated microbubbles adherent to a synthetic vessel coated with avidin increases as much as 20-fold when acoustic radiation force is applied; the adhesion of contrast agents targeted to alpha(v)beta3 expressed on human umbilical vein endothelial cells increases 27-fold within a mimetic vessel when radiation force is applied; and finally, the image signal-to-noise ratio in a phantom vessel increases up to 25 dB using a combination of radiation force and a targeted contrast agent, over use of a targeted contrast agent alone.

State-of-the-art materials for ultrasound-triggered drug delivery.

Ultrasound is a unique and exciting theranostic modality that can be used to track drug carriers, trigger drug release and improve drug deposition with high spatial precision. In this review, we briefly describe the mechanisms of interaction between drug carriers and ultrasound waves, including cavitation, streaming and hyperthermia, and how those interactions can promote drug release and tissue uptake. We then discuss the rational design of some state-of-the-art materials for ultrasound-triggered drug delivery and review recent progress for each drug carrier, focusing on the delivery of chemotherapeutic agents such as doxorubicin. These materials include nanocarrier formulations, such as liposomes and micelles, designed specifically for ultrasound-triggered drug release, as well as microbubbles, microbubble-nanocarrier hybrids, microbubble-seeded hydrogels and phase-change agents.

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.

Advances in Ultrasound Mediated Gene Therapy Using Microbubble Contrast Agents

Microbubble ultrasound contrast agents have the potential to dramatically improve gene therapy treatments by enhancing the delivery of therapeutic DNA to malignant tissue. The physical response of microbubbles in an ultrasound field can mechanically perturb blood vessel walls and cell membranes, enhancing drug permeability into malignant tissue. In this review, we discuss literature that provided evidence of specific mechanisms that enhance in vivo gene delivery utilizing microbubble contrast agents, namely their ability to 1) improving cell membrane permeability, 2) modulate vascular permeability, and 3) enhance endocytotic uptake in cells. Additionally, we review novel microbubble vectors that are being developed in order to exploit these mechanisms and deliver higher gene payloads with greater target specificity. Finally, we discuss some future considerations that should be addressed in the development of next-generation microbubbles in order to improve in vivo microbubble gene delivery. Overall, microbubbles are rapidly gaining popularity as efficient gene carriers, and combined with their functionality as imaging contrast agents, they represent powerful theranostic tools for image guided gene therapy applications.

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.

Ultrasound radiation force modulates ligand availability on targeted contrast agents.

Radiation force produced by low-amplitude ultrasound at clinically relevant frequencies remotely translates freely flowing microbubble ultrasound contrast agents over distances up to centimeters from the luminal space to the vessel wall in order to enhance ligand-receptor contact in targeting applications. The question arises as to how the microbubble shell might be designed at the molecular level to fully take advantage of such physical forces in targeted adhesion for molecular imaging and controlled therapeutic release. Herein, we report on a novel surface architecture in which the tethered ligand is buried in a polymeric overbrush. Our results, with biotin-avidin as the model ligand-receptor pair, show that the overbrush conceals the ligand, thereby reducing immune cell binding and increasing circulation persistence. Targeted adhesion is achieved through application of ultrasound radiation force to instantly reveal the ligand within a well-defined focal zone and simultaneously bind the ligand and receptor. Our data illustrate how the adhesive properties of the contrast agent surface can be reversibly changed, from stealth to sticky, through the physical effects of ultrasound. This technique can be combined with any ligand-receptor pair to optimize targeted adhesion for ultrasonic molecular imaging.