Biomedical Applications for Gas-Stabilizing Solid Cavitation Agents.

For over a decade, advancements in ultrasound-enhanced drug delivery strategies have demonstrated remarkable success in providing targeted drug delivery for a broad range of diseases. In order to achieve enhanced drug delivery, these strategies harness the mechanical effects from bubble oscillations (i.e., cavitation) of a variety of exogenous cavitation agents. Recently, solid cavitation agents have emerged due to their capacity for drug-loading and sustained cavitation duration. Unlike other cavitation agents, solid cavitation agents stabilize gaseous bubbles on hydrophobic surface cavities. Thus, the design of these particles is crucial. In this Review, we provide an overview of the different designs for solid cavitation agents such as nanocups, nanocones, and porous structures, as well as the current status of their development. Considering the numerous advantages of solid cavitation agents, we anticipate further innovations for this new type of cavitation agent across a broad range of biomedical applications.

Ultrasound-Propelled Nanocups for Drug Delivery.

Ultrasound-induced bubble activity (cavitation) has been recently shown to actively transport and improve the distribution of therapeutic agents in tumors. However, existing cavitation-promoting agents are micron-sized and cannot sustain cavitation activity over prolonged time periods because they are rapidly destroyed upon ultrasound exposure. A novel ultrasound-responsive single-cavity polymeric nanoparticle (nanocup) capable of trapping and stabilizing gas against dissolution in the bloodstream is reported. Upon ultrasound exposure at frequencies and intensities achievable with existing diagnostic and therapeutic systems, nanocups initiate and sustain readily detectable cavitation activity for at least four times longer than existing microbubble constructs in an in vivo tumor model. As a proof-of-concept of their ability to enhance the delivery of unmodified therapeutics, intravenously injected nanocups are also found to improve the distribution of a freely circulating IgG mouse antibody when the tumor is exposed to ultrasound. Quantification of the delivery distance and concentration of both the nanocups and coadministered model therapeutic in an in vitro flow phantom shows that the ultrasound-propelled nanocups travel further than the model therapeutic, which is itself delivered to hundreds of microns from the vessel wall. Thus nanocups offer considerable potential for enhanced drug delivery and treatment monitoring in oncological and other biomedical applications.

Investigating the Acoustic Response and Contrast Enhancement of Drug-Loadable PLGA Microparticles with Various Shapes and Morphologies.

Polymer nanoparticles and microparticles have been used primarily for drug delivery. There is now growing interest in further developing polymer-based solid cavitation agents to also enhance ultrasound imaging. We previously reported on a facile method to produce hollow poly(lactic-co-glycolic acid) (PLGA) microparticles with different diameters and degrees of porosity. Here, we investigate the cavitation response from these PLGA microparticles with both therapeutic and diagnostic ultrasound transducers. Interestingly, all formulations exhibited stable cavitation; larger porous and multicavity particles also provided inertial cavitation at elevated acoustic pressure amplitudes. These larger particles also achieved contrast enhancement comparable to that of commercially available ultrasound contrast agents, with a maximum recorded contrast-to-tissue ratio of 28 dB. Therefore, we found that multicavity PLGA microparticles respond to both therapeutic and diagnostic ultrasound and may be applied as a theranostic agent.

Microbubble dissolution in a multigas environment.

Microbubbles occur naturally in the oceans and are used in many industrial and biomedical applications. Here, a theoretical and experimental study was undertaken to determine the fate of a microbubble suddenly suspended in a medium with several gas species as in, for example, the injection of an ultrasound contrast agent into the bloodstream. The model expands on Epstein and Plesset's analysis to include any number of gases. An experimental system was developed which isolates the microbubble in a permeable hollow fiber submerged in a perfusion chamber, allowing rapid exchange of the external aqueous medium. Experimental verification of the model was performed with individual sulfur hexafluoride (SF(6)) microbubbles coated with the soluble surfactant, sodium dodecyl sulfate (SDS). SDS-coated microbubbles suddenly placed in an air-saturated medium initially grew with the influx of O(2) and N(2) and then dissolved under Laplace pressure. SF(6)-filled microbubbles coated with the highly insoluble lipid, dibehenoylphosphatidylcholine, were found to exhibit significantly different behavior owing to a dynamic surface tension. The initial growth phase was diminished, possibly owing to a shell "breakup" tension that exceeded the pure gas/liquid surface tension. Three dissolution regimes were observed: (1) an initial rapid dissolution to the initial diameter followed by (2) steady dissolution with monolayer collapse and finally (3) stabilization below 10 microm diameter. Results indicated that the lipid shell becomes increasingly rigid as the microbubble dissolves, which has important implications on microbubble size distribution, stability, and acoustic properties.

Influence of High Intensity Focused Ultrasound on the Microstructure and c-di-GMP Signaling of Pseudomonas aeruginosa Biofilms.

Bacterial biofilms are typically more tolerant to antimicrobials compared to bacteria in the planktonic phase and therefore require alternative treatment approaches. Mechanical biofilm disruption from ultrasound may be such an alternative by circumventing rapid biofilm adaptation to antimicrobial agents. Although ultrasound facilitates biofilm dispersal and may enhance the effectiveness of antimicrobial agents, the resulting biological response of bacteria within the biofilms remains poorly understood. To address this question, we investigated the microstructural effects of Pseudomonas aeruginosa biofilms exposed to high intensity focused ultrasound (HIFU) at different acoustic pressures and the subsequent biological response. Confocal microscopy images indicated a clear microstructural response at peak negative pressures equal to or greater than 3.5 MPa. In this pressure amplitude range, HIFU partially reduced the biomass of cells and eroded exopolysaccharides from the biofilm. These pressures also elicited a biological response; we observed an increase in a biomarker for biofilm development (cyclic-di-GMP) proportional to ultrasound induced biofilm removal. Cyclic-di-GMP overproducing mutant strains were also more resilient to disruption from HIFU at these pressures. The biological response was further evidenced by an increase in the relative abundance of cyclic-di-GMP overproducing variants present in the biofilm after exposure to HIFU. Our results, therefore, suggest that both physical and biological effects of ultrasound on bacterial biofilms must be considered in future studies.

Author Correction: Remote targeted implantation of sound-sensitive biodegradable multi-cavity microparticles with focused ultrasound.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Remote targeted implantation of sound-sensitive biodegradable multi-cavity microparticles with focused ultrasound.

Ultrasound-enhanced drug delivery has shown great promise in providing targeted burst release of drug at the site of the disease. Yet current solid ultrasound-responsive particles are non-degradable with limited potential for drug-loading. Here, we report on an ultrasound-responsive multi-cavity poly(lactic-co-glycolic acid) microparticle (mcPLGA MP) loaded with rhodamine B (RhB) with or without 4',6-diamidino-2-phenylindole (DAPI) to represent small molecule therapeutics. After exposure to high intensity focused ultrasound (HIFU), these delivery vehicles were remotely implanted into gel and porcine tissue models, where the particles rapidly released their payload within the first day and sustained release for at least seven days. RhB-mcPLGA MPs were implanted with HIFU into and beyond the sub-endothelial space of porcine arteries without observable damage to the artery. HIFU also guided the location of implantation; RhB-mcPLGA MPs were only observed at the focus of the HIFU away from the direction of ultrasound. Once implanted, DAPI co-loaded RhB-mcPLGA MPs released DAPI into the arterial wall, staining the nucleus of the cells. Our work shows the potential for HIFU-guided implantation of drug-loaded particles as a strategy to improve the local and sustained delivery of a therapeutic for up to two weeks.

Exploitation of sub-micron cavitation nuclei to enhance ultrasound-mediated transdermal transport and penetration of vaccines.

Inertial cavitation mediated by ultrasound has been previously shown to enable skin permeabilisation for transdermal drug and vaccine delivery, by sequentially applying the ultrasound then the therapeutic in liquid form on the skin surface. Using a novel hydrogel dosage form, we demonstrate that the use of sub-micron gas-stabilising polymeric nanoparticles (nanocups) to sustain and promote cavitation activity during simultaneous application of both drug and vaccine results in a significant enhancement of both the dose and penetration of a model vaccine, Ovalbumin (OVA), to depths of 500µm into porcine skin. The nanocups themselves exceeded the penetration depth of the vaccine (up to 700µm) due to their small size and capacity to 'self-propel'. In vivo murine studies indicated that nanocup-assisted ultrasound transdermal vaccination achieved significantly (p<0.05) higher delivery doses without visible skin damage compared to the use of a chemical penetration enhancer. Transdermal OVA doses of up to 1µg were achieved in a single 90-second treatment, which was sufficient to trigger an antigen-specific immune response. Furthermore, ultrasound-assisted vaccine delivery in the presence of nanocups demonstrated substantially higher specific anti-OVA IgG antibody levels compared to other transdermal methods. Further optimisation can lead to a viable, safe and non-invasive delivery platform for vaccines with potential use in a primary care setting or personalized self-vaccination at home.

Lipid monolayer collapse and microbubble stability.

Microbubbles are micrometer-size gaseous particles suspended in water, and they are often stabilized by a lipid monolayer shell. Natural microbubbles are found in freshwater and saltwater systems, and engineered microbubbles have a variety of applications in food sciences, biotechnology and medicine. Lipid-coated microbubbles are found to have remarkable stability and mechanical behavior owing to the resistance of the lipid monolayer encapsulation to collapse. The purpose of this review is to tie in recent observations of lipid-coated microbubble dissolution and gas exchange with current literature on the physics of lipid monolayer collapse in the context of lung surfactant. Based on this analysis, we conclude that microbubble shells collapse through the nucleation of microscopic folds, which then catalyze the formation and aggregation of new folds, leading to macroscopic folding events. This process results in a cyclic behavior of crumple-to-smooth transitions, which can be modulated through lipid composition. Eventually, the microbubbles stabilize at 1-2 µm diameter, regardless of initial size or lipid composition, and various mechanisms for this stabilization are postulated. Our ultimate goal is to inspire the reader to consider lipid monolayer collapse as the main long-term stabilizing mechanism for lipid-coated microbubbles, and to stimulate the use of microbubbles as a platform for studying monolayer collapse phenomena.

Theranostic oxygen delivery using ultrasound and microbubbles.

Means to overcome tumor hypoxia have been the subject of clinical investigations since the 1960's; however these studies have yet to find a treatment which is widely accepted. It has been known for nearly a century that hypoxic cells are more resistant to radiotherapy than aerobic cells, and tumor hypoxia is a major factor leading to the resistance of tumors to radiation treatment as well as several cytotoxic agents. In this manuscript, the application of ultrasound combined with oxygen-carrier microbubbles is demonstrated as a method to locally increase dissolved oxygen. Microbubbles can also be imaged by ultrasound, thus providing the opportunity for image-guided oxygen delivery. Simulations of gas diffusion and microbubble gas exchange show that small amounts (down to 5 vol%) of a low-solubility osmotic gas can substantially increase microbubble persistence and therefore production rates and stability of oxygen-carrier microbubbles. Simulations also indicate that the lipid shell can be engineered with long-chain lipids to increase oxygen payload during in vivo transit. Experimental results demonstrate that the application of ultrasound to destroy the microbubbles significantly enhances the local oxygen release. We propose this technology as an application for ultrasound image-guided release of oxygen directly to hypoxic tissue, such as tumor sites to enhance radiotherapy.

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.