Radwa H. Abou-Saleh

Expanding 3D geometry for enhanced on-chip microbubble production and single step formation of liposome modified microbubbles

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.

Nanomechanics of Lipid Encapsulated Microbubbles with Functional Coatings

Microbubbles (MBs) are increasingly being proposed as delivery vehicles for targeted therapeutics, as well as being contrast agents for ultrasound imaging. MBs formed with a lipid shell are promising candidates due to their biocompatibility and the opportunity for surface functionalization, both for specific targeting of tissues and as a means to tune their mechanical response for localized ultrasound induced destruction in vivo. Herein, we acquired force-deformation data on coated lipid MBs using tip-less microcantilevers in an atomic force microscope. Model lipid MBs were designed to test the effects of adding a functional coating on the outside of the lipid leaflet, including a protein coat (streptavidin) or the addition of quantum dots (Q-dots) as optical reporters. MBs (~3 μm diameter) were repeatedly compressed for deformations up to ~50% to obtain a full bubble response. Addition of a coating increased the initial deformation stiffness related to shell bending ~2-fold for streptavidin and ∼3-fold for Q-dots. The presence of a polyethylene glycol (PEG) linker in between the lipid and functional coating, led to enhanced stiffening at high deformations. The plasticity index has been determined and only those MBs that included the PEG linker showed a force dependent short time-scale (<~1s) plasticity. This study demonstrates modulation of the mechanical response of biocompatible MBs through the addition of functional coatings necessary for rationale design of therapeutic lipid MBs for targeted drug delivery.

Poly(ethylene glycol) lipid-shelled microbubbles: abundance, stability, and mechanical properties

Poly(ethylene glycol) (PEG) is widely used on the outside of biomedical delivery vehicles to impart stealth properties. Encapsulated gas microbubbles (MBs) are being increasingly considered as effective carriers for therapeutic intervention to deliver drug payloads or genetic vectors. MBs have the advantage that they can be imaged and manipulated by ultrasound fields with great potential for targeted therapy and diagnostic purposes. Lipid-shelled MBs are biocompatible and can be functionalized on the outer surface for tissue targeting and new therapeutic methods. As MBs become a key route for drug delivery, exploring the effect of PEG-ylation on the MB properties is important. Here, we systematically investigate the effect of PEG-lipid solution concentration ranging between 0 and 35 mol % on the formation of MBs in a microfluidic flow-focusing device. The abundance of the MBs is correlated with the MB lifetime and the whole MB mechanical response, as measured by AFM compression using a tipless cantilever. The maximal MB concentration and stability (lifetime) occurs at a low concentration of PEG-lipid (∼5 mol %). For higher PEG-lipid concentrations, the lifetime and MB concentration decrease, and are accompanied by a correlation between the predicted surface PEG configuration and the whole MB stiffness, as measured at higher compression loads. These results inform the rationale design and fabrication of lipid-based MBs for therapeutic applications and suggest that only relatively small amounts of PEG incorporation are required for optimizing MB abundance and stability while retaining similar mechanical response at low loads.

Research spotlight: microbubbles for therapeutic delivery.

Systemic injection of chemotherapy agents for treating cancer can cause severe side effects for the patient, as well as being a relatively inefficient use of expensive and highly toxic drugs. The area of targeted drug delivery in which drugs are delivered utilizing a specialized carrier directly to the cancerous tumor via immuno-recognition has gained much interest in recent years. Such an approach reduces the side effects of systemic injection and also provides a localized, high-concentration treatment directly to the cancer. Our group at the University of Leeds (Leeds, UK) is developing therapeutic microbubbles that double as both agents for contrast-enhanced ultrasound imaging and drug-delivery vehicles that are targeted to specific cancer cell receptors. Ultimately, a large amplitude sound wave will be used to destroy the bubbles and trigger release of the drug at the targeted tumor. Theranostic microbubbles are a simple and versatile drug-delivery technique that could potentially improve cancer treatment, both in terms of patient experience and overall drug efficiency. Importantly, they offer new ways of delivering hydrophobic drugs, which have traditionally been difficult to deliver efficiently.

Evaluation of Lipid-Stabilised Tripropionin Nanodroplets as a Delivery Route for Combretastatin A4

Lipid-based nanoemulsions are a cheap and elegant route for improving the delivery of hydrophobic drugs. Easy and quick to prepare, nanoemulsions have promise for the delivery of different therapeutic agents. Although multiple studies have investigated the effects of the oil and preparation conditions on the size of the nanoemulsion nanodroplets for food applications, analogous studies for nanoemulsions for therapeutic applications are limited. Here we present a study on the production of lipid-stabilised oil nanodroplets (LONDs) towards medical applications. A number of biocompatible oils were used to form LONDs with phospholipid coatings, and among these, squalane and tripropionin were chosen as model oils for subsequent studies. LONDs were formed by high pressure homogenisation, and their size was found to decrease with increasing production pressure. When produced at 175MPa, all LONDs samples exhibited sizes between 100 and 300nm, with polydispersity index PI between 0.1 and 0.3. The LONDs were stable for over six weeks, at 4°C, and also under physiological conditions, showing modest changes in size (<10%). The hydrophobic drug combretastatin A4 (CA4) was encapsulated in tripropionin LONDs with an efficiency of approximately 76%, achieving drug concentration of approximately 1.3mg/ml. SVR mouse endothelial cells treated with CA4 tripropionin LONDs showed the microtubule disruption, characteristic of drug uptake for all tested doses, which suggests successful release of the CA4 from the LONDs.

High-frequency subharmonic imaging of liposome-loaded microbubbles

The therapeutic application of microbubbles is a highly active area of research, since they can be used for drug or gene delivery. The mouse model is a necessary step in the validation of any new therapeutic agent and high-frequency ultrasound imaging is a common tool used to study this. Attachment of drug-filled liposomes to the shell of a phospholipid microbubble is a common approach for the delivery of a therapeutic payload with ultrasound contrast agents. This study demonstrated that the attachment these drug-filled liposomes had a negligible effect on their response under high frequency ultrasound imaging at 40 MHz and resulted in subharmonic emissions at lower transmit power and greater magnitude in vitro and in vivo.

Interactions between callose and cellulose revealed through the analysis of biopolymer mixtures

The properties of (1,3)-β-glucans (i.e., callose) remain largely unknown despite their importance in plant development and defence. Here we use mixtures of (1,3)-β-glucan and cellulose, in ionic liquid solution and hydrogels, as proxies to understand the physico-mechanical properties of callose. We show that after callose addition the stiffness of cellulose hydrogels is reduced at a greater extent than predicted from the ideal mixing rule (i.e., the weighted average of the individual components’ properties). In contrast, yield behaviour after the elastic limit is more ductile in cellulose-callose hydrogels compared with sudden failure in 100% cellulose hydrogels. The viscoelastic behaviour and the diffusion of the ions in mixed ionic liquid solutions strongly indicate interactions between the polymers. Fourier-transform infrared analysis suggests that these interactions impact cellulose organisation in hydrogels and cell walls. We conclude that polymer interactions alter the properties of callose-cellulose mixtures beyond what it is expected by ideal mixing.Despite their importance in plant development and defence the properties of (1,3)-β-glucan remain largely unknown. Here, the authors find that addition of (1,3)-β-glucans increases the flexibility of cellulose and its resilience to high strain, an effect originating in molecular level interactions.