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Microfluidic system for high throughput characterisation of echogenic particles.

Echogenic particles, such as microbubbles and volatile liquid micro/nano droplets, have shown considerable potential in a variety of clinical diagnostic and therapeutic applications. The accurate prediction of their response to ultrasound excitation is however extremely challenging, and this has hindered the optimisation of techniques such as quantitative ultrasound imaging and targeted drug delivery. Existing characterisation techniques, such as ultra-high speed microscopy provide important insights, but suffer from a number of limitations; most significantly difficulty in obtaining large data sets suitable for statistical analysis and the need to physically constrain the particles, thereby altering their dynamics. Here a microfluidic system is presented that overcomes these challenges to enable the measurement of single echogenic particle response to ultrasound excitation. A co-axial flow focusing device is used to direct a continuous stream of unconstrained particles through the combined focal region of an ultrasound transducer and a laser. Both the optical and acoustic scatter from individual particles are then simultaneously recorded. Calibration of the device and example results for different types of echogenic particle are presented, demonstrating a high throughput of up to 20 particles per second and the ability to resolve changes in particle radius down to 0.1 μm with an uncertainty of less than 3%.

Characterisation of Functionalised Microbubbles for Ultrasound Imaging and Therapy

Liposome production by microfluidics: potential and limiting factors.

This paper provides an analysis of microfluidic techniques for the production of nanoscale lipid-based vesicular systems. In particular we focus on the key issues associated with the microfluidic production of liposomes. These include, but are not limited to, the role of lipid formulation, lipid concentration, residual amount of solvent, production method (including microchannel architecture), and drug loading in determining liposome characteristics. Furthermore, we propose microfluidic architectures for the mass production of liposomes with a view to potential industrial translation of this technology.

Electroformation of Giant Unilamellar Vesicles on Stainless Steel Electrodes.

Giant unilamellar vesicles (GUVs) are well-established model systems for studying membrane structure and dynamics. Electroformation, also referred to as electroswelling, is one of the most prevalent methods for producing GUVs, as it enables modulation of the lipid hydration process to form relatively monodisperse, defect-free vesicles. Currently, however, it is expensive and time-consuming compared with other methods. In this study, we demonstrate that 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine GUVs can be prepared readily at a fraction of the cost on stainless steel electrodes, such as commercially available syringe needles, without any evidence of lipid oxidation or hydrolysis.

Spectral imaging toolbox: segmentation, hyperstack reconstruction, and batch processing of spectral images for the determination of cell and model membrane lipid order.

BACKGROUND: Spectral imaging with polarity-sensitive fluorescent probes enables the quantification of cell and model membrane physical properties, including local hydration, fluidity, and lateral lipid packing, usually characterized by the generalized polarization (GP) parameter. With the development of commercial microscopes equipped with spectral detectors, spectral imaging has become a convenient and powerful technique for measuring GP and other membrane properties. The existing tools for spectral image processing, however, are insufficient for processing the large data sets afforded by this technological advancement, and are unsuitable for processing images acquired with rapidly internalized fluorescent probes. RESULTS: Here we present a MATLAB spectral imaging toolbox with the aim of overcoming these limitations. In addition to common operations, such as the calculation of distributions of GP values, generation of pseudo-colored GP maps, and spectral analysis, a key highlight of this tool is reliable membrane segmentation for probes that are rapidly internalized. Furthermore, handling for hyperstacks, 3D reconstruction and batch processing facilitates analysis of data sets generated by time series, z-stack, and area scan microscope operations. Finally, the object size distribution is determined, which can provide insight into the mechanisms underlying changes in membrane properties and is desirable for e.g. studies involving model membranes and surfactant coated particles. Analysis is demonstrated for cell membranes, cell-derived vesicles, model membranes, and microbubbles with environmentally-sensitive probes Laurdan, carboxyl-modified Laurdan (C-Laurdan), Di-4-ANEPPDHQ, and Di-4-AN(F)EPPTEA (FE), for quantification of the local lateral density of lipids or lipid packing. CONCLUSIONS: The Spectral Imaging Toolbox is a powerful tool for the segmentation and processing of large spectral imaging datasets with a reliable method for membrane segmentation and no ability in programming required. The Spectral Imaging Toolbox can be downloaded from https://uk.mathworks.com/matlabcentral/fileexchange/62617-spectral-imaging-toolbox .

Optimized shapes of magnetic arrays for drug targeting applications

Arrays of permanent magnet elements have been utilized as light-weight, inexpensive sources for applying external magnetic fields in magnetic drug targeting applications, but they are extremely limited in the range of depths over which they can apply useful magnetic forces. In this paper, designs for optimized magnet arrays are presented, which were generated using an optimization routine to maximize the magnetic force available from an arbitrary arrangement of magnetized elements, depending on a set of design parameters including the depth of targeting (up to 50 mm from the magnet) and direction of force required. A method for assembling arrays in practice is considered, quantifying the difficulty of assembly and suggesting a means for easing this difficulty without a significant compromise to the applied field or force. Finite element simulations of in vitro magnetic retention experiments were run to demonstrate the capability of a subset of arrays to retain magnetic microparticles against flow. The results suggest that, depending on the choice of array, a useful proportion of particles (more than 10%) could be retained at flow velocities up to 100 mm/s or to depths as far as 50 mm from the magnet. Finally, the optimization routine was used to generate a design for a Halbach array optimized to deliver magnetic force to a depth of 50 mm inside the brain.

Nanoparticle-Loaded Protein-Polymer Nanodroplets for Improved Stability and Conversion Efficiency in Ultrasound Imaging and Drug Delivery

A new formulation of volatile nanodroplets stabilized by a protein and polymer coating and loaded with magnetic nanoparticles is developed. The droplets show enhanced stability and phase conversion efficiency upon ultrasound exposure compared with existing formulations. Magnetic targeting, encapsulation, and release of an anticancer drug are demonstrated in vitro with a 40% improvement in cytotoxicity compared with free drug.

High-throughput production of microbubble contrast agents using an ultrasound-modulated microfluidic device

We present an ultrasound-modulated microfluidic device for large scale production of microbubble contrast agents. Aim of this device is to overcome limitations associated with bulk production techniques or traditional microfluidic approaches. Microbubbles were characterised in terms of their dimensional and acoustical properties, and compared to those produced using bulk sonication.

A combined magnetic-acoustic device for simultaneous, co-aligned application of magnetic and ultrasonic fields

Acoustically-responsive microbubbles have been widely researched as agents for both diagnostic and therapeutic applications of ultrasound. Recently, there has also been considerable interest in magnetically functionalised microbubbles as multi-modality imaging agents and carriers for magnetically targeted drug delivery. The latter application in particular requires simultaneous application of magnetic and acoustic fields to a target region. This can present a significant practical challenge, especially in vivo where access is typically limited. In this paper, we present a design for an integrated device capable of generating co-aligned magnetic and acoustic fields in order to accumulate microbubbles at a specific location and then to activate them acoustically. For the purposes of this proof of concept study, the magnetic component of the device was designed to concentrate microbubbles at a distance of 10 mm from the probe’s surface, commensurate with relevant tissue depths in preclinical small animal models. The ultrasound transducer was designed to maximise the acoustic intensity in the same region in order to induce cavitation of the magnetically captured microbubbles. Previous studies have indicated that both microbubble concentration and duration of cavitation activity are positively correlated with therapeutic effect. The ability of the device to trap and activate microbubbles was therefore assessed by a series of in vitro tests in a tissue mimicking phantom containing a single vessel of 1.2 mm diameter. At a flow rate of 4.2 mm/s magnetic trapping produced an increase in intensity under B-mode ultrasound imaging consistent with the predicted accumulation profile. When the microbubbles were exposed to the ultrasound field from the probe, the resulting cavitation activity was sustained for a period more than 4 times longer than that achieved with an identical acoustic field but in the absence of a magnet. The feasibility of developing a larger scale device for human applications is discussed.

Layered acoustofluidic resonators for the simultaneous optical and acoustic characterisation of cavitation dynamics, microstreaming, and biological effects.

The study of the effects of ultrasound-induced acoustic cavitation on biological structures is an active field in biomedical research. Of particular interest for therapeutic applications is the ability of oscillating microbubbles to promote both cellular and tissue membrane permeabilisation and to improve the distribution of therapeutic agents in tissue through extravasation and convective transport. The mechanisms that underpin the interaction between cavitating agents and tissues are, however, still poorly understood. One challenge is the practical difficulty involved in performing optical microscopy and acoustic emissions monitoring simultaneously in a biologically compatible environment. Here we present and characterise a microfluidic layered acoustic resonator (μLAR) developed for simultaneous ultrasound exposure, acoustic emissions monitoring, and microscopy of biological samples. The μLAR facilitates in vitro ultrasound experiments in which measurements of microbubble dynamics, microstreaming velocity fields, acoustic emissions, and cell-microbubble interactions can be performed simultaneously. The device and analyses presented provide a means of performing mechanistic in vitro studies that may benefit the design of predictable and effective cavitation-based ultrasound treatments.

Physical Vein Models to Quantify the Flow Performance of Sclerosing Foams.

Foam sclerotherapy is clinically employed to treat varicose veins. It involves intravenous injection of foamed surfactant agents causing endothelial wall damage and vessel shrinkage, leading to subsequent neovascularization. Foam production methods used clinically include manual techniques, such as the Double Syringe System (DSS) and Tessari (TSS) methods. Pre-clinical in-vitro studies are conducted to characterize the performance of sclerosing agents; however, the experimental models used often do not replicate physiologically relevant physical and biological conditions. In this study, physical vein models (PVMs) were developed and employed for the first time to characterize the flow behavior of sclerosing foams. PVMs were fabricated in polydimethylsiloxane (PDMS) by replica molding, and were designed to mimic qualitative geometrical characteristics of veins. Foam behavior was investigated as a function of different physical variables, namely (i) geometry of the vein model (i.e., physiological vs. varicose vein), (ii) foam production technique, and (iii) flow rate of a blood surrogate. The experimental set-up consisted of a PVM positioned on an inclined platform, a syringe pump to control the flow rate of a blood substitute, and a pressure transducer. The static pressure of the blood surrogate at the PVM inlet was measured upon foam administration. The recorded pressure-time curves were analyzed to quantify metrics of foam behavior, with a particular focus on foam expansion and degradation dynamics. Results showed that DSS and TSS foams had similar expansion rate in the physiological PVM, whilst DSS foam had lower expansion rate in the varicose PVM compared to TSS foam. The degradation rate of DSS foam was lower than TSS foam, in both model architectures. Moreover, the background flow rate had a significant effect on foam behavior, enhancing foam displacement rate in both types of PVM.

Early biofilm and streamer formation is mediated by wall shear stress and surface wettability: A multifactorial microfluidic study.

Biofilms are intricate communities of microorganisms encapsulated within a self-produced matrix of extra-polymeric substances (EPS), creating complex three-dimensional structures allowing for liquid and nutrient transport through them. These aggregations offer constituent microorganisms enhanced protection from environmental stimuli-like fluid flow-and are also associated with higher resistance to antimicrobial compounds, providing a persistent cause of concern in numerous sectors like the marine (biofouling and aquaculture), medical (infections and antimicrobial resistance), dentistry (plaque on teeth), food safety, as well as causing energy loss and corrosion. Recent studies have demonstrated that biofilms interact with microplastics, often influencing their pathway to higher trophic levels. Previous research has shown that initial bacterial attachment is affected by surface properties. Using a microfluidic flow cell, we have investigated the relationship between both wall shear stress (τw ) and surface properties (surface wettability) upon biofilm formation of two species (Cobetia marina and Pseudomonas aeruginosa). We investigated biofilm development on low-density polyethylene (LDPE) membranes, Permanox® slides, and glass slides, using nucleic acid staining and end-point confocal laser scanning microscopy. The results show that flow conditions affect biomass, maximum thickness, and surface area of biofilms, with higher τw (5.6 Pa) resulting in thinner biofilms than lower τw (0.2 Pa). In addition, we observed differences in biofilm development across the surfaces tested, with LDPE typically demonstrating more overall biofilm in comparison to Permanox® and glass. Moreover, we demonstrate the formation of biofilm streamers under laminar flow conditions within straight micro-channels.

Potential strategies to prevent encrustations on urinary stents and catheters - thinking outside the box: a European network of multidisciplinary research to improve urinary stents (ENIUS) initiative.

Introduction: Urinary stents have been around for the last 4 decades, urinary catheters even longer. They are associated with infections, encrustation, migration, and patient discomfort. Research efforts to improve them have shifted onto molecular and cellular levels. ENIUS brought together translational scientists to improve urinary implants and reduce morbidity.Methods & materials: A working group within the ENIUS network was tasked with assessing future research lines for the improvement of urinary implants.Topics were researched systematically using Embase and PubMed databases. Clinicaltrials.gov was consulted for ongoing trials.Areas covered: Relevant topics were coatings with antibodies, enzymes, biomimetics, bioactive nano-coats, antisense molecules, and engineered tissue. Further, pH sensors, biodegradable metals, bactericidal bacteriophages, nonpathogenic uropathogens, enhanced ureteric peristalsis, electrical charges, and ultrasound to prevent stent encrustations were addressed.Expert opinion: All research lines addressed in this paper seem viable and promising. Some of them have been around for decades but are yet to proceed to clinical application (i.e. tissue engineering). Others are very recent and, at least in urology, still only conceptual (i.e. antisense molecules). Perhaps the most important learning point resulting from this pan-European multidisciplinary effort is that collaboration between all stakeholders is not only fruitful but also truly essential.

Acoustofluidic device for acoustic capture of Bacillus anthracis spore analogues at low concentration.

A portable device for the rapid concentration of Bacillus subtilis var niger spores, also known as Bacillus globigii (BG), using a thin-reflector acoustofluidic configuration is described. BG spores form an important laboratory analog for the Bacillus anthracis spores, a serious health and bioterrorism risk. Existing systems for spore detection have limitations on detection time and detection that will benefit from the combination with this technology. Thin-reflector acoustofluidic devices can be cheaply and robustly manufactured and provide a more reliable acoustic force than previously explored quarter-wave resonator systems. The system uses the acoustic forces to drive spores carried in sample flows of 30 ml/h toward an antibody functionalized surface, which captures and immobilizes them. In this implementation, spores were fluorescently labeled and imaged. Detection at concentrations of 100 CFU/ml were demonstrated in an assay time of 10 min with 60% capture. We envisage future systems to incorporate more advanced detection of the concentrated spores, leading to rapid, sensitive detection in the presence of significant noise.

Foam-in-Vein: Characterisation of Blood Displacement Efficacy of Liquid Sclerosing Foams.

Sclerotherapy is among the least invasive and most commonly utilised treatment options for varicose veins. Nonetheless, it does not cure varicosities permanently and recurrence rates are of up to 64%. Although sclerosing foams have been extensively characterised with respect to their bench-top properties, such as bubble size distribution and half-life, little is known about their flow behaviour within the venous environment during treatment. Additionally, current methods of foam characterisation do not recapitulate the end-point administration conditions, hindering optimisation of therapeutic efficacy. Here, a therapeutically relevant apparatus has been used to obtain a clinically relevant rheological model of sclerosing foams. This model was then correlated with a therapeutically applicable parameter-i.e., the capability of foams to displace blood within a vein. A pipe viscometry apparatus was employed to obtain a rheological model of 1% polidocanol foams across shear rates of 6 s-1 to 400 s-1. Two different foam formulation techniques (double syringe system and Tessari) and three liquid-to-gas ratios (1:3, 1:4 and 1:5) were investigated. A power-law model was employed on the rheological data to obtain the apparent viscosity of foams. In a separate experiment, a finite volume of foam was injected into a PTFE tube to displace a blood surrogate solution (0.2% w/v carboxymethyl cellulose). The displaced blood surrogate was collected, weighed, and correlated with foam's apparent viscosity. Results showed a decreasing displacement efficacy with foam dryness and injection flowrate. Furthermore, an asymptotic model was formulated that may be used to predict the extent of blood displacement for a given foam formulation and volume. The developed model could guide clinicians in their selection of a foam formulation that exhibits the greatest blood displacement efficacy.

Foam-in-vein: rheological characterisation of liquid sclerosing foams using a pipe viscometer

Sclerotherapy is one of the most common and least-invasive treatment methods for varicose veins. While bench-top properties of sclerosing foams (e.g., bubble size distribution and foam half-life) have been studied previously, their flow behaviour and its relationship to therapeutic efficacy remain largely uncharacterised. To address this research gap, the present study reports on a novel approach for the rheological characterisation of sclerosing foams aimed at obtaining clinically-applicable data. A pipe viscometry apparatus was employed under conditions that mimic the end-point therapeutic application of foams. Polidocanol (1% v/v) foams of various liquid-to-gas volume ratios (1:3, 1:4 and 1:5) were formulated manually using the Tessari and DSS (double syringe system) methods across a clinically-relevant range of shear rates (≈ 7s−1 – 400s−1), in polytetrafluoroethylene pipes of different diameters (2.48 mm and 4.48 mm). Additionally, end-effect and wall-slip correction methods were utilised to model the nominal rheology of sclerosing foams. The rheological data were fitted into a power-law model to obtain fluid flow index (n) and fluid consistency index (K) of sclerosing foams, followed by an in-depth statistical analysis of the power-law indices. The observed rheological behaviour of sclerosing foams is shown to be dependent on vessel diameter and liquid-to-gas ratio, while the type of manual formulation technique used appears to be statistically insignificant towards foam rheology. Sclerosing foams behaved as shear-thinning fluids with observed flow indices ranging 0.238 < n K

Facile production of quercetin nanoparticles using 3D printed centrifugal flow reactors.

Drug nanocrystals are a delivery system comprised of an active pharmaceutical ingredient, with small amounts of a surface stabilizer. Despite offering simplicity in formulation, their manufacture can be a challenging endeavour; this is especially true when the production is performed using microfluidic devices. Although precipitation within microchannels can lead to issues such as clogging, microfluidics is an appealing manufacturing method as it provides fine control over mixing conditions. This allows production of nanoparticles with a narrower size distribution and greater reproducibility compared to batch methods. To generate microfluidic devices cost effectively, replica moulding techniques are considered the manufacturing standard. Due to its simplicity and relatively low cost, 3D printing has become prevalent at the laboratory scale, especially during iterative development of new devices. A challenge of microfluidic-based methods is that they require specialized equipment and multi-step procedures, making them less accessible to users with no previous experience. In a recent study we developed a 3D printed flow-through reactor, referred to as reactor-in-a-centrifuge (RIAC). It is a simple device designed to fit in a 50 mL tube and actuated using a laboratory centrifuge, which removes the need for specialized instrumentation. The manufacturing capabilities of the RIAC have been already proven, by reproducible production of liposomes and silver nanoparticles. The present work demonstrates the use of RIACs with a straight- and spiral-shaped channel architecture to produce quercetin nanocrystals, with therapeutically relevant size (190-302 nm) and very low size dispersity (polydispersity index, PDI < 0.1). The work focused on evaluating how changes in operational parameters (actuation speed) and formulation components (medium viscosity and stabilizer type), impacted on nanocrystal size and PDI. Under all tested conditions the obtained nanocrystals had a smaller size and narrower size distribution, when compared to those produced with alternative methods. The obtained quercetin nanosuspensions however showed limited stability, which should be addressed in future investigations. The simplicity of the RIAC makes it an appealing technology to research groups, especially in low-resource settings and without prior expertise in microfluidics.

Cationic Microbubbles for Non-Selective Binding of Cavitation Nuclei to Bacterial Biofilms.

The presence of multi-drug resistant biofilms in chronic, persistent infections is a major barrier to successful clinical outcomes of therapy. The production of an extracellular matrix is a characteristic of the biofilm phenotype, intrinsically linked to antimicrobial tolerance. The heterogeneity of the extracellular matrix makes it highly dynamic, with substantial differences in composition between biofilms, even in the same species. This variability poses a major challenge in targeting drug delivery systems to biofilms, as there are few elements both suitably conserved and widely expressed across multiple species. However, the presence of extracellular DNA within the extracellular matrix is ubiquitous across species, which alongside bacterial cell components, gives the biofilm its net negative charge. This research aims to develop a means of targeting biofilms to enhance drug delivery by developing a cationic gas-filled microbubble that non-selectively targets the negatively charged biofilm. Cationic and uncharged microbubbles loaded with different gases were formulated and tested to determine their stability, ability to bind to negatively charged artificial substrates, binding strength, and, subsequently, their ability to adhere to biofilms. It was shown that compared to their uncharged counterparts, cationic microbubbles facilitated a significant increase in the number of microbubbles that could both bind and sustain their interaction with biofilms. This work is the first to demonstrate the utility of charged microbubbles for the non-selective targeting of bacterial biofilms, which could be used to significantly enhance stimuli-mediated drug delivery to the bacterial biofilm.

3D printed reactor-in-a-centrifuge (RIAC): making flow-synthesis of nanoparticles pump-free and cost-effective

It is widely recognised that flow-reactors offer greater control over the stoichiometry of chemical reactions when compared to batch methods, since they provide finer and more predictable regulation over the transport of fluids and chemical species. These characteristics are of critical importance in the context of nanoparticle production, since the physical and chemical properties of the fluidic environment within a reactor strongly influence the size and/or shape of the end-product. In the past decade, replica moulding techniques (e.g., based on soft-lithography) have been developed to manufacture flow-reactors in a relatively cost-effective and efficient fashion. However, devices are often operated using multiple syringe pumps, and several of these techniques require laborious and multi-step procedures. In this study, we developed rapidly prototyped reactors embedded within a cylindrical structure that are designed for actuation using a laboratory centrifuge (herein referred to as reactor-in-a-centrifuge, or RIAC). Using RIACs of different architecture, we demonstrated production of nanoscale liposomes of therapeutically relevant size (in the diameter range 80 – 300 nm) under varying operating conditions. We also demonstrated production of silver nanospheres (with UV–vis absorption maxima of 404 nm) at selected operating conditions. The novel concept proposed in this study has the potential to significantly simplify the synthesis of nanomaterials over more commonly used microfluidic techniques, as it relies on a cost-effective and single-step reactor manufacturing process (using a desktop 3D printer) and employs widely available laboratory centrifuges to drive reagents through the reactor. In this paper we describe RIAC's design, manufacturing, and actuation protocols, and demonstrate its applicability to the flow synthesis of nanoparticles without relying on highly specialised instrumentation or costly procedures.

Quantifying ultrasonic deformation of cell membranes with ultra-high-speed imaging

We present a new method for controllable loading of cell models in an ultrasonic (20 kHz) regime. The protocol is based on the inertial-based ultrasonic shaking test and allows to deform cells in the range of few mm/m to help understand potential consequences of repeated loading characteristic of ultrasonic cutting.