Tiny Bubbles….Big Things

The microbubble consortium started in ~2011 bringing together researchers from molecular medicine and physics at the University of Leeds, with an interest in the fabrication of bubbles to enhance the treatment of colorectal cancer.  In particular, we were interested in microbubble production using microfluidic approaches to create monodisperse microbubbles with the size between one and eight microns. However, as part of this, we have developed a patented microfluidic ‘microspray’ technology that produces high concentrations of bubbles that range from a few hundred nanometers to a few microns in diameter. Initially, we used this mixed population of bubbles to develop therapeutic (drug-loaded) microbubbles for localized delivery of drug to tumors. More recently we have started to separate the nanobubble populations by centrifugation to allow us to explore their properties as agents for enhanced localized therapeutic delivery and to understand how the size of the bubbles affects their ability to porate cells and enhance drug uptake.

Microbubbles come in a variety of architectures. Historically, they have found application in contrast-enhanced ultrasound imaging, where the acoustic impedance mismatch between gas and water, as well as non-linear bubble-ultrasound interactions, produces strong scattering of the sound waves. This provides improved contrast in ultrasound imaging. Typically, such bubbles consist of a gas core (usually a fluorocarbon gas) coated with a polymer or phospholipid shell.  Ideally, the gas is chosen to have low solubility in the blood – hence the choice of fluorocarbons.  However, for some purposes using other gases have advantage e.g., oxygen bubbles for enhancing therapeutic action. The protein, lipid, or polymer shell acts as a barrier to reduce gas diffusion and can also be used to enhance biocompatibility and/or introduce molecular targeting.  In therapeutic microbubbles, it is possible to directly attach drugs or drug vehicles (e.g. liposomes) to the bubble surface – to create a theranostic microbubble.

Ultra-Fine Bubbles (UFBs) are similar to microbubbles in terms of their structure but they are of reduced dimension. Such sub-micron bubbles are colloquially referred to as nanobubbles (NBs). As with their larger microbubble counterparts, these nanobubbles are effective scatterers of ultrasound and bring new potential for treatments. Further, it is envisaged that they might offer advantages in being able to better escape the vasculature and penetrate tumors.

In Leeds, we have developed the microfluidic platform “Horizon” for producing micro- / nano-bubbles. It is a ‘plug and play’ platform that allows users to fabricate different types of bubbles, e.g. monodisperse, micro-spray, and nanobubbles by simply switching the microfluidic chip. The platform was first developed to allow reproducible, controlled bubble production across different laboratories and different users within Leeds but the latest version has been shipped to several leading bubble groups across the world. This system is designed to be portable, allowing easy transportation between labs. It is compatible with high-speed imaging to allow fundamental studies of bubble production to be made as well as the production of bubbles for preclinical imaging or therapeutic applications. A key feature of the Horizon is the use of pressure-based pumping to provide pulsatile-free liquid flow. This helps provide better control over the size and dispersity of the bubble populations. This is important for applications where matching the bubble resonance frequency to that of the ultrasound being used for insonation is required, such as sonoporation or disruption of the blood-brain barrier. The 3D expansion built into our patented “spray regime” chips leads to higher bubble concentrations and high-volume production rates. It produces bubbles with an average bubble diameter of ~2um, at concentrations of up to 10^9 MBs/mL in just a few minutes^[1-3]. We have also introduced mF chips that include channels to add therapeutic payload or molecular targeting agents to the bubble surface on-chip which minimizes the off-chip handling of bubbles. These types of therapeutic bubbles are of interest for the treatment of cancer, infection and other diseases.

In our microfluidic production of bubbles, we typically resuspend the lipids of choice (for coating the bubbles) in water. Gas bubbles produced in the aqueous phase are rapidly coated with a phospholipid layer via self-assembly onto the hydrophobic gas/water interface. At the end of this process, our solutions will contain a mixture of bubbles and lipid micelles/vesicles.

Currently, we use three different characterization techniques for our bubbles: Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA), and Resonant Mass Measurement (RMM) supplied by Malvern Panalytical. Microbubbles can be readily separated from lipid particles by floatation. Repeated separation and re-suspension can result in solutions with reduced amounts of lipid particles compared to bubbles. It is important, post-production, to assess the size and concentration of the bubbles (and the other lipid product) remaining in solution. To do this we use a combination of optical microscopy (and image analysis) to provide concentration and sizing, and DLS serves as a check on size distributions and to determine the quality of the product. Nanoparticle Tracking Analysis using the NanoSight NS300 is used to assess the population of lipid particles less than a micron in size – usually present in the sub-natant.

For Nanobubbles it is more challenging to separate contributions of nanobubbles from the presence of liposomes and lipid particles. To do this we have used a combination of Nanoparticle Tracking Analysis (NTA, NanoSight NS300), Resonant Mass Measurement (RMM, Archimedes), and TEM. Nanobubble samples are typically separated from microbubbles by floatation and centrifugation and are diluted a thousand-fold prior to analysis.

NTA offers the most convenient method for the determination of the NB concentration and size, and offers the most useful size range coverage for NBs. The fact that it is rapid and requires little pre-measurement preparation means it is used routinely as part of our MB characterization. RMM (Figure 1b) offers the ability to distinguish between positively buoyant particles (i.e. the ones that are less dense than the solution) and negatively buoyant particles (i.e. denser than the solution). RMM also measures the size and concentration of the positively and negatively buoyant particles. The negatively buoyant population likely consists of a combination of lipid particles that were not converted into bubbles as well as potentially containing PFC droplets, which due to their small size have condensed from a gas into liquid PFC droplets. Additionally, NBs are occasionally imaged using TEM, and their size distribution was analyzed. Comparison between the three measurement techniques used shows agreement between both NTA and TEM results in terms of their respective modal sizes and the population distributions^[7].

The combination of these approaches allows us to explore the distribution of the formulated product across bubbles and particles and to also assess the potential loss of material during handling. It is also possible that NTA can be used to differentiate between liposome/lipid particles and nanobubbles due to the differences in their light scattering behaviors. We are exploring this presently.

By controlling the bubble size and the ultrasound excitation frequency it has been shown that microbubbles can be driven into resonance, whereby the oscillating microbubbles near cell surfaces or cellular junctions can induce cell poration or a breaking of the tight junctions thereby improving drug uptake. Alternatively, bursting the bubbles has also been shown to create a jetting effect that also can porate cells and break such boundaries. Thus, combining MB with ultrasound provides a route for increasing drug uptake and therefore therapeutic delivery.

In Leeds, we are interested in the production of therapeutic microbubbles whereby we attach nanoscale drug delivery vehicles, such as liposomes or lipid-coated oil droplets, to the microbubble shell to create a complex architecture consisting of a microbubble surrounded layer of liposomes and with the functional targeting groups. Such therapeutic bubbles can be injected into the vasculature where the combination of molecular targeting and directed ultrasound has led to enhanced localized delivery, whilst reducing this is systemic exposure.  We are actively pursuing such approaches for the treatment of colorectal cancer metastases in the liver, delivery of oxygen across the blood-brain barrier, and the treatment of biofilms[4,5,6].

An exciting new development is that our microfluidic approach is also able to produce nanobubbles on a length scale from 100 nm to 1 μm. Such nanobubbles also provide strong ultrasound signature in vivo and can be encapsulated within liposomes and provide a drug delivery vehicle that can be acoustically activated to release the liposomal contents. In principle, such complexes will have better penetration into tumors and biofilms due to their smaller sizes^[7].

Nano- and microbubbles have potential across a range of clinical applications from the treatment of cancers; either via the improved delivery of drugs or the (co-)delivery of oxygen to improve radio- and chemo-therapy response.  More widely there are a number of other areas such as the treatment of infection and aiding drug delivery to the brain. 

The combination of ultrasound and bubbles to provide poration of cells will also be significant for the delivery of macromolecules and nanomaterials into cells and avoiding the endocytotic uptake mechanism.

There he heads up a multidisciplinary research and development program working in the life sciences/healthcare area. Malvern Panalytical would like to thank Professor Evans for taking the time to discuss with us his work in the development of novel nanomaterials for in vivo imaging and therapeutic delivery and the use of microbubbles for theranostic applications.