Micron sized, lipid stabilized bubbles of gas are of interest as contrast agents for ultra-sound (US) imaging and increasingly as delivery vehicles for targeted, triggered, therapeutic delivery. Microfluidics provides a reproducible means for microbubble production and surface functionalisation. In this study, microbubbles are generated on chip using flow-focussing microfluidic devices that combine streams of gas and liquid through a nozzle a few microns wide and then subjecting the two phases to a downstream pressure drop. While microfluidics has successfully demonstrated the generation of monodisperse bubble populations, these approaches inherently produce low bubble counts. We introduce a new micro-spray flow regime that generates consistently high bubble concentrations that are more clinically relevant compared to traditional monodisperse bubble populations. Final bubble concentrations produced by the micro-spray regime were up to 10(10) bubbles mL(-1). The technique is shown to be highly reproducible and by using multiplexed chip arrays, the time taken to produce one millilitre of sample containing 10(10) bubbles mL(-1) was ∼10 min. Further, we also demonstrate that it is possible to attach liposomes, loaded with quantum dots (QDs) or fluorescein, in a single step during MBs formation.

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Important aspects in engineering gold nanoparticles for theranostic applications include the control of size, optical properties, cytotoxicity, biodistribution and clearance. In this study,

Micron-sized lipid-stabilised bubbles of heavy gas have been utilised as contrast agents for diagnostic ultrasound (US) imaging for many years. Typically bubbles between 1 and 8 μm in diameter are produced to enhance imaging in US by scattering sound waves more efficiently than surrounding tissue. A potential area of interest for Contrast Enhanced Ultrasound (CEUS) are bubbles with diameters <1 μm or 'nanobubbles.' As bubble diameter decreases, ultrasonic resonant frequency increases, which could lead to an improvement in resolution for high-frequency imaging applications when using nanobubbles. In addition, current US contrast agents are limited by their size to the vasculature in vivo. However, molecular-targeted nanobubbles could penetrate into the extra-vascular space of cancerous tissue providing contrast in regions inaccessible to traditional microbubbles. This paper reports a new microfluidic method for the generation of sub-micron sized lipid stabilised particles containing perfluorocarbon (PFC). The nanoparticles are produced in a unique atomisation-like flow regime at high production rates, in excess of 10(6) particles per s and at high concentration, typically >10(11) particles per mL. The average particle diameter appears to be around 100-200 nm. These particles, suspected of being a mix of liquid and gaseous C4F10 due to Laplace pressure, then phase convert into nanometer sized bubbles on the application of US. In vitro ultrasound characterisation from these nanoparticle populations showed strong backscattering compared to aqueous filled liposomes of a similar size. The nanoparticles were stable upon injection and gave excellent contrast enhancement when used for in vivo imaging, compared to microbubbles with an equivalent shell composition.

Because of their size (1–10 μm), microbubble-based drug delivery agents suffer from confinement to the vasculature, limiting tumor penetration and potentially reducing the drug efficacy. Nanobubbles (NBs) have emerged as promising candidates for ultrasound-triggered drug delivery because of their small size, allowing drug delivery complexes to take advantage of the enhanced permeability and retention effect. In this study, we describe a simple method for production of nested-nanobubbles (Nested-NBs) by encapsulation of NBs (∼100 nm) within drug-loaded liposomes. This method combines the efficient and well-established drug-loading capabilities of liposomes while utilizing NBs as an acoustic trigger for drug release. Encapsulation was characterized using transmission electron microscopy with an encapsulation efficiency of 22 ± 2%. Nested-NBs demonstrated echogenicity using diagnostic B-mode imaging, and acoustic emissions were monitored during high-intensity focused ultrasound (HIFU) in addition to monitoring of model drug release. Results showed that although the encapsulated NBs were destroyed by pulsed HIFU [peak negative pressure (PNP) 1.54–4.83 MPa], signified by loss of echogenicity and detection of inertial cavitation, no model drug release was observed. Changing modality to continuous wave (CW) HIFU produced release across a range of PNPs (2.01–3.90 MPa), likely because of a synergistic effect of mechanical and increased thermal stimuli. Because of this, we predict that our NBs contain a mixed population of both gaseous and liquid core particles, which upon CW HIFU undergo rapid phase conversion, triggering liposomal drug release. This hypothesis was investigated using previously described models to predict the existence of droplets and their phase change potential and the ability of this phase change to induce liposomal drug release.

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Most cancer patients receive chemotherapy at some stage of their treatment which makes improving the efficacy of cytotoxic drugs an ongoing and important goal. Despite large numbers of potent anti-cancer agents being developed, a major obstacle to clinical translation remains the inability to deliver therapeutic doses to a tumor without causing intolerable side effects. To address this problem, there has been intense interest in nanoformulations and targeted delivery to improve cancer outcomes. The aim of this work was to demonstrate how vascular endothelial growth factor receptor 2 (VEGFR2)-targeted, ultrasound-triggered delivery with therapeutic microbubbles (thMBs) could improve the therapeutic range of cytotoxic drugs. Methods: Using a microfluidic microbubble production platform, we generated thMBs comprising VEGFR2-targeted microbubbles with attached liposomal payloads for localised ultrasound-triggered delivery of irinotecan and SN38 in mouse models of colorectal cancer. Intravenous injection into tumor-bearing mice was used to examine targeting efficiency and tumor pharmacodynamics. High-frequency ultrasound and bioluminescent imaging were used to visualise microbubbles in real-time. Tandem mass spectrometry (LC-MS/MS) was used to quantitate intratumoral drug delivery and tissue biodistribution. Finally, 89 Zr PET radiotracing was used to compare biodistribution and tumor accumulation of ultrasound-triggered SN38 thMBs with VEGFR2-targeted SN38 liposomes alone. Results: ThMBs specifically bound VEGFR2 in vitro and significantly improved tumor responses to low dose irinotecan and SN38 in human colorectal cancer xenografts. An ultrasound trigger was essential to achieve the selective effects of thMBs as without it, thMBs failed to extend intratumoral drug delivery or demonstrate enhanced tumor responses. Sensitive LC-MS/MS quantification of drugs and their metabolites demonstrated that thMBs extended drug exposure in tumors but limited exposure in healthy tissues, not exposed to ultrasound, by persistent encapsulation of drug prior to elimination. 89 Zr PET radiotracing showed that the percentage injected dose in tumors achieved with thMBs was twice that of VEGFR2-targeted SN38 liposomes alone. Conclusions: thMBs provide a generic platform for the targeted, ultrasound-triggered delivery of cytotoxic drugs by enhancing tumor responses to low dose drug delivery via combined effects on circulation, tumor drug accumulation and exposure and altered metabolism in normal tissues.

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