Bactericidal Effect of Ultrasound-Responsive Microbubbles and Sub-inhibitory Gentamicin against Pseudomonas aeruginosa Biofilms on Substrates With Differing Acoustic Impedance.

The aim of this research was to explore the interaction between ultrasound-activated microbubbles (MBs) and Pseudomonas aeruginosa biofilms, specifically the effects of MB concentration, ultrasound exposure and substrate properties on bactericidal efficacy. Biofilms were grown using a Centre for Disease Control (CDC) bioreactor on polypropylene or stainless-steel coupons as acoustic analogues for soft and hard tissue, respectively. Biofilms were treated with different concentrations of phospholipid-shelled MBs (107-108 MB/mL), a sub-inhibitory concentration of gentamicin (4 µg/mL) and 1-MHz ultrasound with a continuous or pulsed (100-kHz pulse repetition frequency, 25% duty cycle, 0.5-MPa peak-to-peak pressure) wave. The effect of repeated ultrasound exposure with intervals of either 15- or 60-min was also investigated. With polypropylene coupons, the greatest bactericidal effect was achieved with 2 × 5 min of pulsed ultrasound separated by 60 min and a microbubble concentration of 5 × 107 MBs/mL. A 0.76 log (83%) additional reduction in the number of bacteria was achieved compared with the use of an antibiotic alone. With stainless-steel coupons, a 67% (0.46 log) reduction was obtained under the same exposure conditions, possibly due to enhancement of a standing wave field which inhibited MB penetration in the biofilm. These findings demonstrate the importance of treatment parameter selection in antimicrobial applications of MBs and ultrasound in different tissue environments.

Engineering multi-layered tissue constructs using acoustic levitation.

Engineering tissue structures that mimic those found in vivo remains a challenge for modern biology. We demonstrate a new technique for engineering composite structures of cells comprising layers of heterogeneous cell types. An acoustofluidic bioreactor is used to assemble epithelial cells into a sheet-like structure. On transferring these cell sheets to a confluent layer of fibroblasts, the epithelial cells cover the fibroblast surface by collective migration maintaining distinct epithelial and fibroblast cell layers. The collective behaviour of the epithelium is dependent on the formation of cell-cell junctions during levitation and contrasts with the behaviour of mono-dispersed epithelial cells where cell-matrix interactions dominate and hinder formation of discrete cell layers. The multilayered tissue model is shown to form a polarised epithelial barrier and respond to apical challenge. The method is useful for engineering a wide range of layered tissue types and mechanistic studies on collective cell migration.

Generation of functional hepatocyte 3D discoids in an acoustofluidic bioreactor.

Ultrasonic standing wave systems have previously been used for the generation of 3D constructs for a range of cell types. In the present study, we cultured cells from the human hepatoma Huh7 cell line in a Bulk Acoustic Wave field and studied their viability, their functions, and their response to the anti-cancer drug, 5 Fluorouracil (5FU). We found that cells grown in the acoustofluidic bioreactor (AFB) expressed no reduction in viability up to 6 h of exposure compared to those cultured in a conventional 2D system. In addition, constructs created in the AFB and subsequently cultured outside of it had improved functionality including higher albumin and urea production than 2D or pellet cultures. The viability of Huh7 cells grown in the ultrasound field to 5FU anti-cancer drug was comparable to that of cells cultured in the 2D system, showing rapid diffusion into the aggregate core. We have shown that AFB formed 3D cell constructs have improved functionality over the conventional 2D monolayer and could be a promising model for anti-cancer drug testing.

Review of the Development of Methods for Characterization of Microspheres for Use in Embolotherapy: Translating Bench to Cathlab.

Therapeutic embolotherapy is the deliberate occlusion of a blood vessel within the body, which can be for the prevention of internal bleeding, stemming of flow through an arteriovenous malformation, or occlusion of blood vessels feeding a tumor. This is achieved using a wide selection of embolic devices such as balloons, coils, gels, glues, and particles. Particulate embolization is often favored for blocking smaller vessels, particularly within hypervascularized tumors, as they are available in calibrated sizes and can be delivered distally via microcatheters for precise occlusion with associated locoregional drug delivery. Embolic performance has been traditionally evaluated using animal models, but with increasing interest in the 3R's (replacement, reduction, refinement), manufacturers, regulators, and clinicians have shown interest in the development of more sophisticated in vitro methods for evaluation and prediction of in vivo performance. Herein the current progress in developing bespoke techniques incorporating physical handling, fluid dynamics, occlusive behavior, and sustained drug elution kinetics within vascular systems is reviewed. While it is necessary to continue to validate the safety of such devices in vivo, great strides have been made in the development of bench tests that better predict the behavior of these products aligned with the principles of the 3R's.

Impact of Yttrium-90 Microsphere Density, Flow Dynamics, and Administration Technique on Spatial Distribution: Analysis Using an In Vitro Model.

PURPOSE: To investigate material density, flow, and viscosity effects on microsphere distribution within an in vitro model designed to simulate hepatic arteries. MATERIALS AND METHODS: A vascular flow model was used to compare distribution of glass and resin surrogates in a clinically derived flow range (60-120 mL/min). Blood-mimicking fluid (BMF) composed of glycerol and water (20%-50% vol/vol) was used to simulate a range of blood viscosities. Microsphere distribution was quantified gravimetrically, and injectate solution was dyed to enable quantification by UV spectrophotometry. Microsphere injection rate (5-30 mL/min) and the influence of contrast agent dilution of injection solution (0%-60% vol/vol) were also investigated. RESULTS: No significant differences in behavior were observed between the glass and resin surrogate materials under any tested flow conditions (P = .182; n = 144 injections). Microspheres tend to align more consistently with the saline injection solution (r2 = 0.5712; n = 144) compared with total BMF flow distribution (r2 = 0.0104; n = 144). The most predictable injectate distribution (ie, greatest alignment with BMF flow, < 5% variation) was demonstrated with > 10-mL/min injection rates of pure saline solution, although < 20% variation with glass microsphere distribution was observed with injection solution containing as much as 30% contrast medium when injected at > 20 mL/min. CONCLUSIONS: Glass and resin yttrium-90 surrogates demonstrated similar distribution in a range of clinically relevant flow conditions, suggesting that microsphere density does not have a significant influence on microsphere distribution. Injection parameters that enhanced the mixing of the spheres with the BMF resulted in the most predictable distribution.

Spatiotemporal dynamics of doxorubicin elution from embolic beads within a microfluidic network.

Anticancer treatment using embolic drug-eluting beads (DEBs) has shown multifarious advantages compared to systemic chemotherapy. However, there is a growing need for a better understanding of the physical parameters governing drug-elution from embolic devices under physiologically relevant fluidic conditions. In the present study, we investigated the spatiotemporal dynamics of doxorubicin hydrochloride elution from drug-loaded hydrogel embolic beads within a microfluidic device consisting of a network of interconnected microchannels which replicates the architectural properties of microvascular systems. Drug-elution has been investigated experimentally at a single-bead level, using in-house developed microscopy- and spectrofluorimetry-based methods. Results demonstrated that the kinetics of drug-elution and the amount of eluted drug strongly depended on the location of the embolic event within the embolised channel (e.g. fractional amount of eluted drug after 3h was equal to ~0.2 and ~0.6 for completely-confined and partially-confined bead, respectively). Drug-elution from partially-confined bead showed a counterintuitive dependence on the local Reynolds number (and thus on the mean fluid velocity), as a result of dynamic changes in bead compressibility causing the displacement of the bead from the primary embolic site. Conversely, the kinetics of drug-elution from fully-confined bead was less affected by the local Reynolds number and bead displayed faster elution from the surface area exposed to the systemic flow, which was associated with the formation of fluid eddies nearby the bead post embolisation.

High throughput imaging cytometer with acoustic focussing.

We demonstrate an imaging flow cytometer that uses acoustic levitation to assemble cells and other particles into a sheet structure. This technique enables a high resolution, low noise CMOS camera to capture images of thousands of cells with each frame. While ultrasonic focussing has previously been demonstrated for 1D cytometry systems, extending the technology to a planar, much higher throughput format and integrating imaging is non-trivial, and represents a significant jump forward in capability, leading to diagnostic possibilities not achievable with current systems. A galvo mirror is used to track the images of the moving cells permitting exposure times of 10 ms at frame rates of 50 fps with motion blur of only a few pixels. At 80 fps, we demonstrate a throughput of 208 000 beads per second. We investigate the factors affecting motion blur and throughput, and demonstrate the system with fluorescent beads, leukaemia cells and a chondrocyte cell line. Cells require more time to reach the acoustic focus than beads, resulting in lower throughputs; however a longer device would remove this constraint.

Deformation of red blood cells using acoustic radiation forces.

Acoustic radiation forces have been used to manipulate cells and bacteria in a number of recent microfluidic applications. The net force on a cell has been subject to careful investigation over a number of decades. We demonstrate that the radiation forces also act to deformcells. An ultrasonic standing wave field is created in a 0.1 mm glass capillary at a frequency of 7.9 MHz. Using osmotically swollen red-blood cells, we show observable deformations up to an aspect ratio of 1.35, comparable to deformations created by optical tweezing. In contrast to optical technologies, ultrasonic devices are potentially capable of deforming thousands of cells simultaneously. We create a finite element model that includes both the acoustic environment of the cell, and a model of the cell membrane subject to forces resulting from the non-linear aspects of the acoustic field. The model is found to give reasonable agreement with the experimental results, and shows that the deformation is the result of variation in an acoustic force that is directed outwards at all points on the cell membrane. We foresee applications in diagnostic devices, and in the possibility of mechanically stimulating cells to promote differentiation and physiological effects.

Efficient finite element modeling of radiation forces on elastic particles of arbitrary size and geometry.

A finite element based method is presented for calculating the acoustic radiation force on arbitrarily shaped elastic and fluid particles. Importantly for future applications, this development will permit the modeling of acoustic forces on complex structures such as biological cells, and the interactions between them and other bodies. The model is based on a non-viscous approximation, allowing the results from an efficient, numerical, linear scattering model to provide the basis for the second-order forces. Simulation times are of the order of a few seconds for an axi-symmetric structure. The model is verified against a range of existing analytical solutions (typical accuracy better than 0.1%), including those for cylinders, elastic spheres that are of significant size compared to the acoustic wavelength, and spheroidal particles.

Mithramycin encapsulated in polymeric micelles by microfluidic technology as novel therapeutic protocol for beta-thalassemia.

This report shows that the DNA-binding drug, mithramycin, can be efficiently encapsulated in polymeric micelles (PM-MTH), based on Pluronic(®) block copolymers, by a new microfluidic approach. The effect of different production parameters has been investigated for their effect on PM-MTH characteristics. The compared analysis of PM-MTH produced by microfluidic and conventional bulk mixing procedures revealed that microfluidics provides a useful platform for the production of PM-MTH with improved controllability, reproducibility, smaller size, and polydispersity. Finally, an investigation of the effects of PM-MTH, produced by microfluidic and conventional bulk mixing procedures, on the erythroid differentiation of both human erythroleukemia and human erythroid precursor cells is reported. It is demonstrated that PM-MTH exhibited a slightly lower toxicity and more pronounced differentiative activity when compared to the free drug. In addition, PM-MTH were able to upregulate preferentially γ-globin messenger ribonucleic acid production and to increase fetal hemoglobin (HbF) accumulation, the percentage of HbF-containing cells, and their HbF content without stimulating α-globin gene expression, which is responsible for the clinical symptoms of ß-thalassemia. These results represent an important first step toward a potential clinical application, since an increase in HbF could alleviate the symptoms underlying ß-thalassemia and sickle cell anemia. In conclusion, this report suggests that PM-MTH produced by microfluidic approach warrants further evaluation as a potential therapeutic protocol for ß-thalassemia.

The selection of layer thicknesses to control acoustic radiation force profiles in layered resonators.

Ultrasonic standing waves can be used to generate radiation forces on particles within a fluid. A number of authors have derived detailed representations of these forces but these are most commonly applied using an approximation to the energy distribution based upon an idealized standing wave within a mode based upon rigid boundaries. An electro-acoustic model of the acoustic energy distribution within a standing wave with arbitrary thickness boundaries has been expanded to model the radiation force on an example particle within the acoustic field. This is used to examine the force profile on a particle at resonances other than those predicted with rigid boundaries, and with pressure nodes at different positions. A simple analytical method for predicting modal conditions for combinations of frequencies and layer thickness characteristics is presented, which predicts that resonances can exist that will produce a pressure node at arbitrary positions in the fluid layer of such a system. This can be used to design resonators that will drive particles to positions other than the center of the fluid layer, including the fluid/solid boundary of the layer, with significant potential applications in sensing systems. Further, the model also predicts conditions for multiple subwavelength resonances within the fluid layer of a single resonator, each resonance having different nodal planes for particle concentration.