A theoretical investigation of photoacoustic contrast agents.

Photoacoustic imaging offers significant potential as a biomedical imaging modality. For some applications, however, there is a need for contrast enhancement. In this paper, a theoretical comparison is presented of the efficacy of three different designs for photoacoustic contrast agents (PACAs), specifically, a droplet of dye, a bubble filled with gas coated by a dye loaded shell, and a droplet of volatile dye. For each case, the governing equations describing the dynamics of a single PACA in a homogenous incompressible fluid are derived. The coupled sets of equations describing the bubble oscillation and resulting radiated pressure, the photo-acoustic energy equation, and the equation of state are then solved numerically. The numerical results predict a stronger radiated acoustic signal for the same optical source energy density in the case of the volatile dye droplet by a factor of up to two orders of magnitude compared with the other two types of agent.

Electrospinning versus fibre production methods: from specifics to technological convergence.

Academic and industrial research on nanofibres is an area of increasing global interest, as seen in the continuously multiplying number of research papers and patents and the broadening range of chemical, medical, electrical and environmental applications. This in turn expands the size of the market opportunity and is reflected in the significant rise of entrepreneurial activities and investments in the field. Electrospinning is probably the most researched top-down method to form nanofibres from a remarkable range of organic and inorganic materials. It is well known and discussed in many comprehensive studies, so why this review? As we read about yet another "novel" method producing multifunctional nanomaterials in grams or milligrams in the laboratory, there is hardly any research addressing how these methods can be safely, consistently and cost-effectively up-scaled. Despite two decades of governmental and private investment, the productivity of nanofibre forming methods is still struggling to meet the increasing demand. This hinders the further integration of nanofibres into practical large-scale applications and limits current uses to niche-markets. Looking into history, this large gap between supply and demand of synthetic fibres was seen and addressed in conventional textile production a century ago. The remarkable achievement was accomplished via extensive collaborative research between academia and industry, applying ingenious solutions and technological convergence from polymer chemistry, physical chemistry, materials science and engineering disciplines. Looking into the present, current advances in electrospinning and nanofibre production are showing similar interdisciplinary technological convergence, and knowledge of industrial textile processing is being combined with new developments in nanofibre forming methods. Moreover, many important parameters in electrospinning and nanofibre spinning methods overlap parameters extensively studied in industrial fibre processing. Thus, this review combines interdisciplinary knowledge from the academia and industry to facilitate technological convergence and offers insight for upscaling electrospinning and nanofibre production. It will examine advances in electrospinning within a framework of large-scale fibre production as well as alternative nanofibre forming methods, providing a comprehensive comparison of conventional and contemporary fibre forming technologies. This study intends to stimulate interest in addressing the issue of scale-up alongside novel developments and applications in nanofibre research.

An investigation into the cytotoxic effects of microbubbles and their constituents on osteosarcoma and bone marrow stromal cells.

BACKGROUND: Ultrasound-responsive microbubbles offer a means of achieving minimally invasive, localised drug delivery in applications including regenerative medicine. To facilitate their use, however, it is important to determine any cytotoxic effects they or their constituents may have. The aim of this study was to test the hypothesis that phospholipid-shelled microbubbles are non-toxic to human bone-derived cells at biologically-relevant concentrations. METHODS: Microbubbles were fabricated using combinations of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC), polyoxyethylene(40) stearate (PEG40S) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene-glycol)-2000] (DSPE-PEG2000). Microbubble size and concentration were measured as a function of time and temperature by optical microscopy. Effects on MG63 osteosarcoma and human bone marrow stromal cells (BMSCs) were measured for up to 72 h by assay for viability, metabolic activity and proliferation. RESULTS: DBPC:DSPE-PEG2000 microbubbles were significantly more stable than DSPC:PEG40S microbubbles under all conditions tested. Serum-containing medium had no detrimental effect on microbubble stability, but storage at 37 °C compared to at 4 °C reduced stability for both preparations, with almost complete dissolution of microbubbles at times ≥24 h. DSPC:PEG40S microbubbles had greater inhibitory effects on cell metabolism and growth than DBPC:DSPE-PEG2000 microbubbles, with PEG40S found to be the principle inhibitory component. These effects were only evident at high microbubble concentrations (≥20% (v/v)) or with prolonged culture (≥24 h). Increasing cell-microbubble contact by inversion culture in a custom-built device had no inhibitory effect on metabolism. CONCLUSIONS: These data indicate that, over a broad range of concentrations and incubation times, DBPC:DSPE-PEG2000 and DSPC:PEG40S microbubbles have little effect on osteoblastic cell viability and growth, and that PEG40S is the principle inhibitory component in the formulations investigated.

Cavitation and contrast: the use of bubbles in ultrasound imaging and therapy.

Microbubbles and cavitation are playing an increasingly significant role in both diagnostic and therapeutic applications of ultrasound. Microbubble ultrasound contrast agents have been in clinical use now for more than two decades, stimulating the development of a range of new contrast-specific imaging techniques which offer substantial benefits in echocardiography, microcirculatory imaging, and more recently, quantitative and molecular imaging. In drug delivery and gene therapy, microbubbles are being investigated/developed as vehicles which can be loaded with the required therapeutic agent, traced to the target site using diagnostic ultrasound, and then destroyed with ultrasound of higher intensity energy burst to release the material locally, thus avoiding side effects associated with systemic administration, e.g. of toxic chemotherapy. It has moreover been shown that the motion of the microbubbles increases the permeability of both individual cell membranes and the endothelium, thus enhancing therapeutic uptake, and can locally increase the activity of drugs by enhancing their transport across biologically inaccessible interfaces such as blood clots or solid tumours. In high-intensity focused ultrasound (HIFU) surgery and lithotripsy, controlled cavitation is being investigated as a means of increasing the speed and efficacy of the treatment. The aim of this paper is both to describe the key features of the physical behaviour of acoustically driven bubbles which underlie their effectiveness in biomedical applications and to review the current state of the art.

Enhanced efficacy in drug-resistant cancer cells through synergistic nanoparticle mediated delivery of cisplatin and decitabine.

There are several limitations with monodrug cancer therapy, including poor bioavailability, rapid clearance and drug resistance. Combination therapy addresses these by exploiting synergism between different drugs against cancer cells. In particular, the combination of epigenetic therapies with conventional chemotherapeutic agents can improve the initial tumour response and overcome acquired drug resistance. Co-encapsulation of multiple therapeutic agents into a single polymeric nanoparticle is one of the many approaches taken to enhance therapeutic effect and improve the pharmacokinetic profile. In this study, different types of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), matrix and core-shell (CS), were investigated for simultaneous encapsulation of a demethylating drug, decitabine, and a potent anticancer agent, cisplatin. It was shown that by altering the configuration of the CS structure, the release profile could be tuned. In order to investigate whether this could enhance the anticancer effect compared to cisplatin, human ovarian carcinoma cell line (A2780) and its cisplatin resistant variant (A2780cis) were exposed to free cisplatin and the CS-NPs. A better response was obtained in both cell lines (11% and 51% viability of A2780 and A2780cis, respectively) using CS-NPs than cisplatin alone (27%, 82% viability of A2780 and A2780cis, respectively) or in combination with decitabine (22%, 96% viability of A2780 and A2780cis, respectively) at equivalent doses (10 µM).

Preparation of suspensions of phospholipid-coated microbubbles by coaxial electrohydrodynamic atomization.

The use of phospholipid-coated microbubbles for medical applications is gaining considerable attention. However, the preparation of lipid-coated microbubble suspensions containing the ideal size and size distribution of bubbles still represents a considerable challenge. The most commonly used preparation methods of sonication and mechanical agitation result in the generation of polydisperse microbubbles with diameters ranging from less than 1 microm to greater than 50 microm. Efforts have been made via distinctly different techniques such as microfluidic and electrohydrodynamic bubbling to prepare lipid-coated microbubbles with diameters less than 10 microm and with a narrow size distribution, and recent results have been highly promising. In this paper, we describe a detailed investigation of the latter method that essentially combines liquid and air flow, and an applied electric field to generate microbubbles. A parametric plot was constructed between the air flow rate (Qg) and the lipid suspension flow rate (Ql) to identify suitable flow rate regimes for the preparation of phospholipid-coated microbubbles with a mean diameter of 6.6 microm and a standard deviation of 2.5 microm. The parametric plot has also helped in developing a scaling equation between the bubble diameter and the ratio Qg/Ql. At ambient temperature (22 degrees C), these bubbles were very stable with their size remaining almost unchanged for 160 min. The influence of higher temperatures such as the human body temperature (37 degrees C) on the size and stability of the microbubbles was also explored. It was found that the mean bubble diameter fell rapidly to begin with but then stabilized at 1-2 microm after 20 min.

Ultrasound-activated microbubbles as a novel intracellular drug delivery system for urinary tract infection.

The development of new modalities for high-efficiency intracellular drug delivery is a priority for a number of disease areas. One such area is urinary tract infection (UTI), which is one of the most common infectious diseases globally and which imposes an immense economic and healthcare burden. Common uropathogenic bacteria have been shown to invade the urothelial wall during acute UTI, forming latent intracellular reservoirs that can evade antimicrobials and the immune response. This behaviour likely facilitates the high recurrence rates after oral antibiotic treatments, which are not able to penetrate the bladder wall and accumulate to an effective concentration. Meanwhile, oral antibiotics may also exacerbate antimicrobial resistance and cause systemic side effects. Using a human urothelial organoid model, we tested the ability of novel ultrasound-activated lipid microbubbles to deliver drugs into the cytoplasm of apical cells. The gas-filled lipid microbubbles were decorated with liposomes containing the non-cell-permeant antibiotic gentamicin and a fluorescent marker. The microbubble suspension was added to buffer at the apical surface of the bladder model before being exposed to ultrasound (1.1 MHz, 2.5 Mpa, 5500 cycles at 20 ms pulse duration) for 20 s. Our results show that ultrasound-activated intracellular delivery using microbubbles was over 16 times greater than the control group and twice that achieved by liposomes that were not associated with microbubbles. Moreover, no cell damage was detected. Together, our data show that ultrasound-activated microbubbles can safely deliver high concentrations of drugs into urothelial cells, and have the potential to be a more efficacious alternative to traditional oral antibiotic regimes for UTI. This modality of intracellular drug delivery may prove useful in other clinical indications, such as cancer and gene therapy, where such penetration would aid in treatment.

Sonoprinting liposomes on tumor spheroids by microbubbles and ultrasound.

Ultrasound-triggered drug-loaded microbubbles have great potential for drug delivery due to their ability to locally release drugs and simultaneously enhance their delivery into the target tissue. We have recently shown that upon applying ultrasound, nanoparticle-loaded microbubbles can deposit nanoparticles onto cells grown in 2D monolayers, through a process that we termed "sonoprinting". However, the rigid surfaces on which cell monolayers are typically growing might be a source of acoustic reflections and aspherical microbubble oscillations, which can influence microbubble-cell interactions. In the present study, we aim to reveal whether sonoprinting can also occur in more complex and physiologically relevant tissues, by using free-floating 3D tumor spheroids as a tissue model. We show that both monospheroids (consisting of tumor cells alone) and cospheroids (consisting of tumor cells and fibroblasts, which produce an extracellular matrix) can be sonoprinted. Using doxorubicin-liposome-loaded microbubbles, we show that sonoprinting allows to deposit large amounts of doxorubicin-containing liposomes to the outer cell layers of the spheroids, followed by doxorubicin release into the deeper layers of the spheroids, resulting in a significant reduction in cell viability. Sonoprinting may become an attractive approach to deposit drug patches at the surface of tissues, thereby promoting the delivery of drugs into target tissues.

Increasing the nonlinear character of microbubble oscillations at low acoustic pressures.

The nonlinear response of gas bubbles to acoustic excitation is an important phenomenon in both the biomedical and engineering sciences. In medical ultrasound imaging, for example, microbubbles are used as contrast agents on account of their ability to scatter ultrasound nonlinearly. Increasing the degree of nonlinearity, however, normally requires an increase in the amplitude of excitation, which may also result in violent behaviour such as inertial cavitation and bubble fragmentation. These effects may be highly undesirable, particularly in biomedical applications, and the aim of this work was to investigate alternative means of enhancing nonlinear behaviour. In this preliminary report, it is shown through theoretical simulation and experimental verification that depositing nanoparticles on the surface of a bubble increases the nonlinear character of its response significantly at low excitation amplitudes. This is due to the fact that close packing of the nanoparticles restricts bubble compression.

Generation of multilayered structures for biomedical applications using a novel tri-needle coaxial device and electrohydrodynamic flow.

In this short communication, we describe the scope and flexibility of using a novel device containing three coaxially arranged needles to form a variety of novel morphologies. Different combinations of materials are subjected to controlled flow through the device under the influence of an applied electric field. The resulting electrohydrodynamic flow allows us to prepare double-layered bubbles, porous encapsulated threads and nanocapsules containing three layers. The ability to process such multilayered structures is very significant for biomedical engineering applications, for example, generating capsules for drug delivery, which can provide multistage controlled release.

Novel co-axial electrohydrodynamic in-situ preparation of liquid-filled polymer-shell microspheres for biomedical applications.

Suspensions consisting of polymer-shelled microspheres are finding increasing use in a diverse range of technologies or applications, e.g. in the medical field, such as diagnostic imaging, drug and gene delivery and tissue engineering. In this work, a solution of water-insoluble polymethylsilsesquioxane was perfused through the outer needle of a co-axial needle arrangement while air was passed simultaneously through the inner needle, with both needles placed in an electric field. The liquid and air flow rates were varied but at 5 microl s(-1) for each material stable microbubble formation was achieved at 5.7 kV. The microbubbles were collected in a vial of distilled water and they rapidly converted into polymer-shelled microspheres containing approximately 60 wt% liquid. Microscopic examination of the spheres within 300 s of preparation showed a large population of near-spherical polymer-shelled microspheres with a mean size of 6 +/- 2 microm diameter near the water surface. After 48 h, the microspheres had collected at the bottom of the vial. The fact that the microspheres absorbed and encapsulated the liquid in which they were collected and the fact that their size (< 10 microm) is suitable for vascular administration make this a new one-step preparation technology for microspheres used in biomedical applications.

Preparation of polymeric and ceramic porous capsules by a novel electrohydrodynamic process.

The preparation of capsules for medical and industrial use can be achieved via several conventional routes, yielding either hard or soft receptacles, depending on the type and the content of the material to be encapsulated. Together with tablets, capsules are amongst the most commonly used means of administering medication and this makes progress in capsule preparation technology a key area of drug delivery research. Here we uncover new technology for the preparation of capsules with porous chambers. The novelty is signified in the use of an electrohydrodynamic process engineering route and its potential is elucidated using a polymeric material; polymethylsilsesquioxane, which can be converted into an identical ceramic form by means of simple pyrolysis. Thus, both polymeric and ceramic capsules have been prepared. The effects of process control parameters such as the applied voltage and flow rate, on the characteristics of the capsules prepared are 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.

Microbubbling by co-axial electrohydrodynamic atomization.

The preparation of microbubble suspensions is an important feature of medical engineering research. Recently, co-axial electrohydrodynamic atomization was used in our laboratory for the first time to prepare microbubble suspensions. In this paper, using a model glycerol-air system, we investigate in detail the characteristics of this microbubbling process. Modes of microbubbling are elucidated with respect to applied voltage and liquid and air flow rates. Thus, a parametric plot is constructed to identify a liquid and gas flow rate regime, which allows continuous microbubbling. This map provides a basis for the selection of a suitable combination of liquid and gas flow rates particularly in relation to yield and bubble size. The mechanism of microbubbling in microfluidic systems is compared with that of microbubbling by co-axial electrohydrodynamic atomization to identify the advantages and the limiting factors of the latter. Stability of microbubbles prepared by this method in terms of variation of diameter as a function of time is compared with previous literature on the dissolution of microbubbles with an air core and suggests the need for further work to stabilize the bubbles.

Preparation of microbubble suspensions by co-axial electrohydrodynamic atomization.

In this paper we report a novel method, based on co-axial electrohydrodynamic jetting, for the preparation of microbubble suspensions containing bubbles <10 microm in size and having a narrow size distribution. No selective filtration is necessary and the suspensions are produced directly by the process. To demonstrate the method, glycerol was used as the liquid medium, flowing in the outer needle of the co-axial twin needle arrangement and undergoing electrohydrodynamic atomization in the stable cone-jet mode while air flowed through the inner needle at the same time. At zero applied voltage a hollow stream of liquid flowed from the outer needle. When the applied voltage was increased, eventually the hollow stream became a stable cone-jet and emitted a microthread of bubbles, which were collected in a container of glycerol to obtain microbubble suspensions. The size of the microbubbles was measured via optical microscopy and laser diffractometry. Several microbubble suspensions were prepared and characterised and the size distribution was found to be critically dependent on the ratio (n) of flow rates of liquid/air and, in particular the flow rate of the air. At n=1.5, with the flow rate of air set at approximately 1.7 microl/s, a microbubble suspension containing bubbles in the size range 2-8 microm was obtained.

Quantification of cell-bubble interactions in a 3D engineered tissue phantom.

Understanding cell-bubble interactions is crucial for preventing bubble related pathologies and harnessing their potential therapeutic benefits. Bubbles can occur in the body as a result of therapeutic intravenous administration, surgery, infections or decompression. Subsequent interactions with living cells, may result in pathological responses such as decompression sickness (DCS). This work investigates the interactions that occur between bubbles formed during decompression and cells in a 3D engineered tissue phantom. Increasing the tissue phantoms' cellular density resulted in decreased dissolved O2 (DO) concentrations (p = 0.0003) measured using real-time O2 monitoring. Direct microscopic observation of these phantoms, revealed a significant (p = 0.0024) corresponding reduction in bubble nucleation. No significant difference in growth rate or maximum size of the bubbles was measured (p = 0.99 and 0.23). These results show that bubble nucleation is dominated by DO concentration (affected by cellular metabolism), rather than potential nucleation sites provided by cell-surfaces. Consequent bubble growth depends not only on DO concentration but also on competition for dissolved gas. Cell death was found to significantly increase (p = 0.0116) following a bubble-forming decompression. By comparison to 2D experiments; the more biomimetic 3D geometry and extracellular matrix in this work, provide data more applicable for understanding and developing models of in vivo bubble dynamics.

A combined three-dimensional in vitro-in silico approach to modelling bubble dynamics in decompression sickness.

The growth of bubbles within the body is widely believed to be the cause of decompression sickness (DCS). Dive computer algorithms that aim to prevent DCS by mathematically modelling bubble dynamics and tissue gas kinetics are challenging to validate. This is due to lack of understanding regarding the mechanism(s) leading from bubble formation to DCS. In this work, a biomimetic in vitro tissue phantom and a three-dimensional computational model, comprising a hyperelastic strain-energy density function to model tissue elasticity, were combined to investigate key areas of bubble dynamics. A sensitivity analysis indicated that the diffusion coefficient was the most influential material parameter. Comparison of computational and experimental data revealed the bubble surface's diffusion coefficient to be 30 times smaller than that in the bulk tissue and dependent on the bubble's surface area. The initial size, size distribution and proximity of bubbles within the tissue phantom were also shown to influence their subsequent dynamics highlighting the importance of modelling bubble nucleation and bubble-bubble interactions in order to develop more accurate dive algorithms.

Magnetic targeting to enhance microbubble delivery in an occluded microarterial bifurcation.

Ultrasound and microbubbles have been shown to accelerate the breakdown of blood clots both in vitro and in vivo. Clinical translation of this technology is still limited, however, in part by inefficient microbubble delivery to the thrombus. This study examines the obstacles to delivery posed by fluid dynamic conditions in occluded vasculature and investigates whether magnetic targeting can improve microbubble delivery. A 2D computational fluid dynamic model of a fully occluded Y-shaped microarterial bifurcation was developed to determine: (i) the fluid dynamic field in the vessel with inlet velocities from 1-100 mm s-1 (corresponding to Reynolds numbers 0.25-25); (ii) the transport dynamics of fibrinolytic drugs; and (iii) the flow behavior of microbubbles with diameters in the clinically-relevant range (0.6-5 µm). In vitro experiments were carried out in a custom-built microfluidic device. The flow field was characterized using tracer particles, and fibrinolytic drug transport was assessed using fluorescence microscopy. Lipid-shelled magnetic microbubbles were fluorescently labelled to determine their spatial distribution within the microvascular model. In both the simulations and experiments, the formation of laminar vortices and an abrupt reduction of fluid velocity were observed in the occluded branch of the bifurcation, severely limiting drug transport towards the occlusion. In the absence of a magnetic field, no microbubbles reached the occlusion, remaining trapped in the first vortex, within 350 µm from the bifurcation center. The number of microbubbles trapped within the vortex decreased as the inlet velocity increased, but was independent of microbubble size. Application of a magnetic field (magnetic flux density of 76 mT, magnetic flux density gradient of 10.90 T m-1 at the centre of the bifurcation) enabled delivery of microbubbles to the occlusion and the number of microbubbles delivered increased with bubble size and with decreasing inlet velocity.

Ultrasound-induced inertial cavitation from gas-stabilizing nanoparticles.

The understanding of cavitation from nanoparticles has been hindered by the inability to control nanobubble size. We present a method to manufacture nanoparticles with a tunable single hemispherical depression (nanocups) of mean diameter 90, 260, or 650 nm entrapping a nanobubble. A modified Rayleigh-Plesset crevice model predicts the inertial cavitation threshold as a function of cavity size and frequency, and is verified experimentally. The ability to tune cavitation nanonuclei and predict their behavior will be useful for applications ranging from cancer therapy to ultrasonic cleaning.

Evaluation of microbubble contrast agents for dynamic imaging with x-ray phase contrast.

X-rays are commonly used as a means to image the inside of objects opaque to visible light, as their short wavelength allows penetration through matter and the formation of high spatial resolution images. This physical effect has found particular importance in medicine where x-ray based imaging is routinely used as a diagnostic tool. Increasingly, however, imaging modalities that provide functional as well as morphological information are required. In this study the potential to use x-ray phase based imaging as a functional modality through the use of microbubbles that can be targeted to specific biological processes is explored. We show that the concentration of a microbubble suspension can be monitored quantitatively whilst in flow using x-ray phase contrast imaging. This could provide the basis for a dynamic imaging technique that combines the tissue penetration, spatial resolution, and high contrast of x-ray phase based imaging with the functional information offered by targeted imaging modalities.