A comparative analysis of low intensity ultrasound effects on living cells: from simulation to experiments.

Ultrasounds are already broadly exploited in clinical diagnostics and are now becoming a powerful and not harmful tool in antitumoral therapies, as they are able to produce damages towards cancer cells, thank to inertial cavitation and temperature increase. The use of US alone or combined to molecular compounds, microbubbles or solid-state nanoparticles is the focus of current research and clinical trials, like thermoablation, drug sonoporation or sonodynamic therapies. In the present work, we discuss on the non-thermal effects of ultrasound and the conditions which enable oxygen radical production and which role they can have in provoking the death of different cancer cell lines. In this perspective, we set a mathematical model to predict the pressure spatial distribution in a defined water sample volume and thus obtain a map of acoustic pressures and acoustic intensities of the applied ultrasound at different input powers. We then validate and verify these numerical results with direct acoustic measurements and by detecting the production of reactive oxygen species (ROS) by means of sonochemiluminescence (SCL) and electron paramagnetic resonance (EPR) spectroscopy, applied to the same water sample volume and using the same US input parameters adopted in the simulation. Finally, the various US conditions are applied to two different set of cancer cell lines, a cervical adenocarcinoma and a hematological cancer, Burkitt's lymphoma. We hypothesize how the ROS generation can influence the recorded cell death. In a second set of experiments, the role of semiconductor metal oxide nanocrystals, i.e. zinc oxide, is also evaluated by adding them to the water and biological systems. In particular, the role of ZnO in enhancing the ROS production is verified. Furthermore, the interplay among US and ZnO nanocrystals is evaluated in provoking cancer cell death at specific conditions. This study demonstrates a useful correlation between numerical simulation and experimental acoustic validation as well as with ROS measurement at both qualitative and quantitative levels during US irradiation of simple water solution. It further tries to translate the obtained results to justify one of the possible mechanisms responsible of cancer cell death. It thus aims to pave the way for the use of US in cancer therapy and a better understanding on the non-thermal effect that a specific set of US parameters can have on cancer cells cultured in vitro.

Nanoparticle-assisted ultrasound: A special focus on sonodynamic therapy against cancer.

At present, ultrasound radiation is broadly employed in medicine for both diagnostic and therapeutic purposes at various frequencies and intensities. In this review article, we focus on therapeutically-active nanoparticles (NPs) when stimulated by ultrasound. We first introduce the different ultrasound-based therapies with special attention to the techniques involved in the oncological field, then we summarize the different NPs used, ranging from soft materials, like liposomes or micro/nano-bubbles, to metal and metal oxide NPs. We therefore focus on the sonodynamic therapy and on the possible working mechanisms under debate of NPs-assisted sonodynamic treatments. We support the idea that various, complex and synergistics physical-chemical processes take place during acoustic cavitation and NP activation. Different mechanisms are therefore responsible for the final cancer cell death and strongly depends not only on the type and structure of NPs or nanocarriers, but also on the way they interact with the ultrasonic pressure waves. We conclude with a brief overview of the clinical applications of the various ultrasound therapies and the related use of NPs-assisted ultrasound in clinics, showing that this very innovative and promising approach is however still at its infancy in the clinical cancer treatment.

Functionalized ZnO nanowires for microcantilever biosensors with enhanced binding capability.

An efficient way to increase the binding capability of microcantilever biosensors is here demonstrated by growing zinc oxide nanowires (ZnO NWs) on their active surface. A comprehensive evaluation of the chemical compatibility of ZnO NWs brought to the definition of an innovative functionalization method able to guarantee the proper immobilization of biomolecules on the nanostructured surface. A noteworthy higher amount of grafted molecules was evidenced with colorimetric assays on ZnO NWs-coated devices, in comparison with functionalized and activated silicon flat samples. ZnO NWs grown on silicon microcantilever arrays and activated with the proposed immobilization strategy enhanced the sensor binding capability (and thus the dynamic range) of nearly 1 order of magnitude, with respect to the commonly employed flat functionalized silicon devices. Graphical Abstract An efficient way to increase the binding capability of microcantilever biosensors is represented by growing zinc oxide nanowires (ZnO NWs) on their active surface. ZnO NWs grown on silicon microcantilever arrays and activated with an innovative immobilization strategy enhanced the sensor binding capability of nearly 1 order of magnitude, with respect to the commonly employed flat functionalized silicon devices.

Impedance characterization, degradation, and in vitro biocompatibility for platinum electrodes on BioMEMS.

Fine control of molecular transport through microfluidic systems can be obtained by modulation of an applied electrical field across channels with the use of electrodes. In BioMEMS designed for biological fluids and in vivo applications, electrodes must be biocompatible, biorobust and stable. In this work, the analysis and characterization of platinum (Pt) electrodes integrated on silicon substrates for biomedical applications are presented. Electrodes were incorporated on the surface of silicon chips by adhesion of laminated Pt foils or deposited at 30°, 45° or 90° angle by e-beam or physical vapor (sputtering) methods. Electrical and physical properties of the electrodes were quantified and evaluated using electrical impedance spectroscopy and modelling of the electrode-electrolyte interfaces. Electrode degradation in saline solution at pH 7.4 was tested at room temperature and under accelerated conditions (90 °C), both in the presence and absence of an applied electrical potential. Degradation was quantified using atomic force microscopy (AFM) and inductively coupled plasma mass spectroscopy (ICP-MS). Biocompatibility was assessed by MTT proliferation assay with human dermal fibroblasts. Results demonstrated that the deposited electrodes were biocompatible with negligible material degradation and exhibited electrochemical behavior similar to Pt foils, especially for e-beam deposited electrodes. Finally, Pt electrodes e-beam deposited on silicon nanofabricated nanochannel membranes were evaluated for controlled drug delivery applications. By tuning a low applied electrical potential (<1.5 VDC) to the electrodes, temporal modulation of the dendritic fullerene 1 (DF-1) release from a source reservoir was successfully achieved as a proof of concept, highlighting the potential of deposited electrodes in biomedical applications.

Flexible tactile sensing based on piezoresistive composites: a review.

The large expansion of the robotic field in the last decades has created a growing interest in the research and development of tactile sensing solutions for robot hand and body integration. Piezoresistive composites are one of the most widely employed materials for this purpose, combining simple and low cost preparation with high flexibility and conformability to surfaces, low power consumption, and the use of simple read-out electronics. This work provides a review on the different type of composite materials, classified according to the conduction mechanism and analyzing the physics behind it. In particular piezoresistors, strain gauges, percolative and quantum tunnelling devices are reviewed here, with a perspective overview on the most used filler types and polymeric matrices. A description of the state-of-the-art of the tactile sensor solutions from the point of view of the architecture, the design and the performance is also reviewed, with a perspective outlook on the main promising applications.

Length-dependent charge generation from vertical arrays of high-aspect-ratio ZnO nanowires.

Aqueous chemical growth of zinc oxide nanowires is a flexible and effective approach to obtain dense arrays of vertically oriented nanostructures with high aspect ratio. Herein we present a systematic study of the different synthesis parameters that influence the ZnO seed layer and thus the resulting morphological features of the free-standing vertically oriented ZnO nanowires. We obtained a homogeneous coverage of transparent conductive substrates with high-aspect-ratio nanowire arrays (length/diameter ratio of up to 52). Such nanostructured vertical arrays were examined to assess their electric and piezoelectric properties, and showed an electric charge generation upon mechanical compressive stress. The principle of energy harvesting with these nanostructured ZnO arrays was demonstrated by connecting them to an electronic charge amplifier and storing the generated charge in a series of capacitors. We found that the generated charge and the electrical behavior of the ZnO nanowires are strictly dependent on the nanowire length. We have shown the importance of controlling the morphological properties of such ZnO nanostructures for optimizing a nanogenerator device.

Insight into Sonoluminescence Augmented by ZnO-Functionalized Nanoparticles.

Recent advances in optical imaging techniques rely on the use of nanosized contrast agents for in vitro and in vivo applications. We report on an imaging method based on the inertial cavitation of ultrasound-irradiated water solutions that lead to sonoluminescence (SL), here, newly proposed in combination with semiconductor nanoparticles, in particular, aminopropyl-functionalized zinc oxide nanocrystals. The obtained measurements confirm the ability of such nanocrystals to increase the sonoluminescence emission, together with the ability to modify the SL spectrum when compared to the pure water behavior. In particular, it is shown that the UV component of SL is absorbed by the semiconductor behavior that is also confirmed in different biologically relevant media. Finally, optical images of nanocrystal-assisted SL are acquired for the first time, in particular, in biological buffers, revealing that at low ultrasound intensities, SL is measurable only when the nanocrystals are present in solution. All of these results witness the role of amine-functionalized zinc oxide nanocrystals for sonoluminescence emission, which makes them very good candidates as efficient nanocontrast agents for SL imaging for biological and biomedical applications.

Effect of process parameters on separation efficiency in a deterministic lateral displacement device.

Deterministic lateral displacement (DLD) is a hydrodynamic method known for its high-resolution sorting of particles. It achieves this through a periodic array of obstacles and laminar flow that passively directs particles along in two different directions depending on the particles' diameter. Many prior publications have been dedicated to the structural and geometrical development of DLD arrays to improve separation performance; however, a successful separation requires much more than a well-designed array. This paper shows how separation performance is affected by process parameters. For this purpose, the design and fabrication of a DLD device are described. Then three experiments show how process parameters affect the performance of the device. The first experiment uses dye solutions to visualize the formation of a hydrodynamically focused sample stream. The second experiment shows that the particle separation performance (of 7- & 15-µm particles) is affected by the way output fluids are collected. Finally, the third experiment looks at the particle separation efficiency as the input flow rates and the ratio of buffer to sample are changed. The results show that the proper range for buffer and sample flow rate in this device is 1-10 and 0.1-1 (µl/min), respectively. The buffer to sample flow rate ratio of 10 gives the highest separation efficiency, but at a lower sample throughput. The optimized values are specific for our device but demonstrate processes that we believe are universal for DLD separations.

A multilevel Lab on chip platform for DNA analysis.

Lab-on-chips (LOCs) are critical systems that have been introduced to speed up and reduce the cost of traditional, laborious and extensive analyses in biological and biomedical fields. These ambitious and challenging issues ask for multi-disciplinary competences that range from engineering to biology. Starting from the aim to integrate microarray technology and microfluidic devices, a complex multilevel analysis platform has been designed, fabricated and tested (All rights reserved-IT Patent number TO2009A000915). This LOC successfully manages to interface microfluidic channels with standard DNA microarray glass slides, in order to implement a complete biological protocol. Typical Micro Electro Mechanical Systems (MEMS) materials and process technologies were employed. A silicon/glass microfluidic chip and a Polydimethylsiloxane (PDMS) reaction chamber were fabricated and interfaced with a standard microarray glass slide. In order to have a high disposable system all micro-elements were passive and an external apparatus provided fluidic driving and thermal control. The major microfluidic and handling problems were investigated and innovative solutions were found. Finally, an entirely automated DNA hybridization protocol was successfully tested with a significant reduction in analysis time and reagent consumption with respect to a conventional protocol.

Focalization Performance Study of a Novel Bulk Acoustic Wave Device.

This work illustrates focalization performances of a silicon-based bulk acoustic wave device applied for the separation of specimens owing to micrometric dimensions. Samples are separated in the microfluidic channel by the presence of an acoustic field, which focalizes particles or cells according to their mechanical properties compared to the surrounded medium ones. Design and fabrication processes are reported, followed by focalization performance tests conducted either with synthetic particles or cells. High focalization performances occurred at different microparticle concentrations. In addition, preliminary tests carried out with HL-60 cells highlighted an optimal separation performance at a high flow rate and when cells are mixed with micro and nanoparticles without affecting device focalization capabilities. These encouraging results showed how this bulk acoustic wave device could be exploited to develop a diagnostic tool for early diagnosis or some specific target therapies by separating different kinds of cells or biomarkers possessing different mechanical properties such as shapes, sizes and densities.

Silicon Carbide-Gated Nanofluidic Membrane for Active Control of Electrokinetic Ionic Transport.

Manipulation of ions and molecules by external control at the nanoscale is highly relevant to biomedical applications. We report a biocompatible electrode-embedded nanofluidic channel membrane designed for electrofluidic applications such as ionic field-effect transistors for implantable drug-delivery systems. Our nanofluidic membrane includes a polysilicon electrode electrically isolated by amorphous silicon carbide (a-SiC). The nanochannel gating performance was experimentally investigated based on the current-voltage (I-V) characteristics, leakage current, and power consumption in potassium chloride (KCl) electrolyte. We observed significant modulation of ionic diffusive transport of both positively and negatively charged ions under physical confinement of nanochannels, with low power consumption. To study the physical mechanism associated with the gating performance, we performed electrochemical impedance spectroscopy. The results showed that the flat band voltage and density of states were significantly low. In light of its remarkable performance in terms of ionic modulation and low power consumption, this new biocompatible nanofluidic membrane could lead to a new class of silicon implantable nanofluidic systems for tunable drug delivery and personalized medicine.

Mesoporous Materials for Drug Delivery and Theranostics.

Mesoporous materials, especially those made of silica or silicon, are capturing great interest in the field of nanomedicine [...].

Piezo- and Photocatalytic Activity of Ferroelectric ZnO:Sb Thin Films for the Efficient Degradation of Rhodamine-ß dye Pollutant.

The discovery of novel catalytic materials showing unprecedented properties and improved functionalities represents a major challenge to design advanced oxidation processes for wastewater purification. In this work, antimony (Sb) doping is proposed as a powerful approach for enhancing the photo- and piezocatalytic performances of piezoelectric zinc oxide (ZnO) thin films. To investigate the role played by the dopant, the degradation of Rhodamine-ß (Rh-ß), a dye pollutant widely present in natural water sources, is studied when the catalyst is irradiated by ultraviolet (UV) light or ultrasound (US) waves. Depending on the doping level, the structural, optical, and ferroelectric properties of the catalyst can be properly set to maximize the dye degradation efficiency. Independently of the irradiation source, the fastest and complete dye degradation is observed in the presence of the doped catalyst and for an optimal amount of the inserted dopant. Among ZnO:Sb samples, the most doped one (5 at. %) shows improved UV light absorption and photocatalytic properties. Conversely, the piezocatalytic efficiency is maximized using the lowest Sb amount (1 at. %). The superior ferroelectric polarization observed in this case highly favors the adsorption of electrically charged species, in particular of the dye in the protonated form (Rh-ß+) and of OH-, to the catalyst surface and the production of hydroxyl radicals responsible for dye degradation.

Leveraging re-chargeable nanobubbles on amine-functionalized ZnO nanocrystals for sustained ultrasound cavitation towards echographic imaging.

Nanoparticles able to promote inertial cavitation when exposed to focused ultrasound have recently gained much attention due to their vast range of possible applications in the biomedical field, such as enhancing drug penetration in tumor or supporting ultrasound contrast imaging. Due to their nanometric size, these contrast agents could penetrate through the endothelial cells of the vasculature to target tissues, thus enabling higher imaging resolutions than commercial gas-filled microbubbles. Herein, Zinc Oxide NanoCrystals (ZnO NCs), opportunely functionalized with amino-propyl groups, are developed as novel nanoscale contrast agents that are able, for the first time, to induce a repeatedly and over-time sustained inertial cavitation as well as ultrasound contrast imaging. The mechanism behind this phenomenon is investigated, revealing that re-adsorption of air gas nanobubbles on the nanocrystal surface is the key factor for this re-chargeable cavitation. Moreover, inertial cavitation and significant echographic signals are obtained at physiologically relevant ultrasound conditions (MI < 1.9), showing great potential for low side-effects in in-vivo applications of the novel nanoscale agent from diagnostic imaging to gas-generating theranostic nanoplatforms and to drug delivery.

Zinc Oxide Nanocrystals and High-Energy Shock Waves: A New Synergy for the Treatment of Cancer Cells.

In the last years, different nanotools have been developed to fight cancer cells. They could be administered alone, exploiting their intrinsic toxicity, or remotely activated to achieve cell death. In the latter case, ultrasound (US) has been recently proposed to stimulate some nanomaterials because of the US outstanding property of deep tissue penetration and the possibility of focusing. In this study, for the first time, we report on the highly efficient killing capability of amino-propyl functionalized ZnO nanocrystals (ZnO NCs) in synergy with high-energy ultrasound shock waves (SW) for the treatment of cancer cells. The cytotoxicity and internalization of ZnO NCs were evaluated in cervical adenocarcinoma KB cells, as well as the safety of the SW treatment alone. Then, the remarkably high cytotoxic combination of ZnO NCs and SW was demonstrated, comparing the effect of multiple (3 times/day) SW treatments toward a single one, highlighting that multiple treatments are necessary to achieve efficient cell death. At last, preliminary tests to understand the mechanism of the observed synergistic effect were carried out, correlating the nanomaterial surface chemistry to the specific type of stimulus used. The obtained results can thus pave the way for a novel nanomedicine treatment, based on the synergistic effect of nanocrystals combined with highly intense mechanical pressure waves, offering high efficiency, deep and focused tissue penetration, and a reduction of side effects on healthy cells.

The Synergistic Effect of Nanocrystals Combined With Ultrasound in the Generation of Reactive Oxygen Species for Biomedical Applications.

Reactive oxygen species (ROS) effects on living cells and tissues is multifaceted and their level or dose can considerably affect cell proliferation and viability. It is therefore necessary understand their role also designing ways able to regulate their amount inside cells, i.e., using engineered nanomaterials with either antioxidant properties or, for cancer therapy applications, capable to induce oxidative stress and cell death, through tunable ROS production. In this paper, we report on the use of single-crystalline zinc oxide (ZnO) round-shaped nanoparticles, yet ZnO nanocrystals (NCs) functionalized with amino-propyl groups (ZnO-NH2 NCs), combined with pulsed ultrasound (US). We show the synergistic effects produced by NC-assisted US which are able to produce different amount of ROS, as a result of inertial cavitation under the pulsed US exposure. Using Passive Cavitation Detection (PCD) and Electron Paramagnetic Resonance (EPR) spectroscopy, we systematically study which are the key parameters, monitoring, and influencing the amount of generated ROS measuring their concentration in water media and comparing all the results with pure water batches. We thus propose a ROS generation mechanism based on the selective application of US to the ZnO nanocrystals in water solutions. Ultrasound B-mode imaging is also applied, proving in respect to pure water, the enhanced ecographic signal generation of the aqueous solution containing ZnO-NH2 NCs when exposed to pulsed ultrasound. Furthermore, to evaluate the applicability of ZnO-NH2 NCs in the biomedical field, the ROS generation is studied by interposing different tissue mimicking materials, like phantoms and ex vivo tissues, between the US transducer and the sample well. As a whole, we clearly proof the enhanced capability to produce ROS and to control their amount when using ZnO-NH2 NCs in combination with pulsed ultrasound anticipating their applicability in the fields of biology and health care.

Sonophotocatalytic degradation mechanisms of Rhodamine B dye via radicals generation by micro- and nano-particles of ZnO.

In this work, it is proposed an environmental friendly sonophotocatalytic approach to efficiently treat polluted waters from industrial dyes exploiting ZnO micro- and nano-materials. For the first time, we deeply investigated the generation of reactive oxygen species (ROS) under ultrasound stimulation of different ZnO structures by Electron Paramagnetic Resonance Spectroscopy (EPR). Indeed, five zinc oxide (ZnO) micro- and nano-structures, i.e. Desert Roses (DRs), Multipods (MPs), Microwires (MWs), Nanoparticles (NPs) and Nanowires (NWs), were studied for the Rhodamine B (RhB) sonodegradation under ultrasonic irradiation. The DRs microparticles demonstrated the best sonocatalytic performance (100% degradation of RhB in 180 min) and the highest OH· radicals generation under ultrasonic irradiation. Strikingly, the coupling of ultrasound and sun-light irradiation in a sonophotodegradation approach led to 100% degradation efficiency, i.e. color reduction, of RhB in just 10 min, revealing a great positive synergy between the photocatalytic and sonocatalytic mechanisms. The RhB sonophotocatalytic degradation was also evaluated at different initial dye concentrations and with the presence of anions in solution. It was demonstrated a good stability over repeated cycles of dye treatment, which probe the applicability of this technique with industrial effluents. In conclusion, sonophotocatalytic degradation synergizing sunlight and ultrasound in the presence of DRs microparticles shows a great potential and a starting point to investigate further the efficient treatment of organic dyes in wastewater.

Graphene Oxide Finely Tunes the Bioactivity and Drug Delivery of Mesoporous ZnO Scaffolds.

Mesoporous zinc oxide (ZnO) scaffolds coated with drop-cast graphene oxide (GO) flakes are proposed to be a novel bilayer system featuring bioactivity, biocompatibility, and promising loading/release properties for controlled drug-delivery systems. The high-surface-area ZnO scaffolds show clear apatite deposition, but their particular surface chemistry and topography prevent the formation of a continuous coating, resulting in micrometric crystalline apatite aggregates after 28 days in simulated body fluid (SBF). When gentamicin sulfate (GS) is considered as a model molecule, pure ZnO scaffolds also show functional GS loading efficiency, with fast in vitro release kinetics driven by a simple diffusion mechanism. Strikingly, the bioactivity and GS delivery properties of mesoporous ZnO are efficiently triggered by drop-casting GO flakes atop the mesoporous scaffold surface. The resulting ZnO@GO bilayer scaffolds show the formation of a uniform apatite coating after 28 days in SBF and demonstrate a biocompatible behavior, supporting the culture of SaOS-2 osteoblast-like cells. Moreover, the GO coating also leads to a barrier-layer effect, preventing fast GS release, particularly in the short time range. This barrier effect, coupled with the existence of nanopores within the GO structure, sieves drug molecules from the mesoporous ZnO matrix and allows for a delayed release of the GS molecule. We, thus, demonstrated a new-generation ZnO@GO bilayer system as effective multifunctional and biocompatible scaffold for bone tissue engineering.

Investigation of Fatigue Behavior of ABS and PC-ABS Polymers at Different Temperatures.

Plastics are widely used in structural components where cyclic loads may cause fatigue failure. In particular, in some applications such as in vehicles, the working temperature may change and therefore the strength of the polymeric materials. In this work, the fatigue behavior of two thermoplastic materials (ABS and PC-ABS) at different temperatures has been investigated. In particular, three temperatures have been considered representing the working condition at room temperature, at low temperature (winter conditions), and high temperature (summer conditions and/or components close to the engine). Results show that high temperature have big impact on fatigue performance, while low temperatures may also have a slight positive effect.

Unexpected behaviors in molecular transport through size-controlled nanochannels down to the ultra-nanoscale.

Ionic transport through nanofluidic systems is a problem of fundamental interest in transport physics and has broad relevance in desalination, fuel cells, batteries, filtration, and drug delivery. When the dimension of the fluidic system approaches the size of molecules in solution, fluid properties are not homogeneous and a departure in behavior is observed with respect to continuum-based theories. Here we present a systematic study of the transport of charged and neutral small molecules in an ideal nanofluidic platform with precise channels from the sub-microscale to the ultra-nanoscale (<5 nm). Surprisingly, we find that diffusive transport of nano-confined neutral molecules matches that of charged molecules, as though the former carry an effective charge. Further, approaching the ultra-nanoscale molecular diffusivities suddenly drop by up to an order of magnitude for all molecules, irrespective of their electric charge. New theoretical investigations will be required to shed light onto these intriguing results.