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Abstract

Hypersound (ultrasound of gigahertz (GHz) frequency) has been recently introduced as a new type of membrane-disruption method for cells, vesicles and supported lipid bilayers (SLBs), with the potential to improve the efficiency of drug and gene delivery for biomedical applications. Here, we fabricated an integrated microchip, composed of a nano-electromechanical system (NEMS) resonator and a gold electrode as the extended gate of a field effect transistor (EGFET), to study the effects of hypersonic poration on an SLB in real time. The current recordings revealed that hypersound enabled ion conduction through the SLB by inducing transient nanopores in the membrane, which act as the equivalent of ion channels and show gating behavior. The mechanism of pore formation was studied by cyclic voltammetry (CV), atomic force microscopy (AFM) and laser scanning microscopy (LSM), which support the causality between hypersound-triggered deformation and the reversible membrane disruption of the SLB. This finding contributes to the development of an approach to reversibly control membrane permeability by hypersound.

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... Cyclic voltammetry, atomic force microscopy, and laser scanning microscopy were used together to characterize the nanopores. 111 The relationship between membrane deformation and poration induced by hypersound was carefully studied. As shown in Fig. 5e, SLB was covered on the resonator, and the gold electrode connected with FET. ...
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IntroductionSonoelectrochemical Production of HydrogenSonochemical Production of Noble Metals and Fuel Cell ElectrocatalystsSonoelectrochemical Production of Noble Metals and Fuel Cell ElectrocatalystsSonochemical and Sonoelectrochemical Preparation of Fuel Cell ElectrodesIndustrial Applications of the Use of Ultrasound for the Fabrication of Fuel Cell MaterialsConclusions AcknowledgementList of AbbreviationsReferences
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Scitation is the online home of leading journals and conference proceedings from AIP Publishing and AIP Member Societies
Article
DOI:https://doi.org/10.1103/PhysRevLett.2.298
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Article
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The development of oriented organic monomolecular layers by the Langmuir-Blodgett (LB) and self-assembly (SA) techniques has led researchers toward their goal of assembling individual molecules into highly ordered architectures. Thus the continually growing contribution of LB and SA systems to the chemistry and physics of thin organic films is widely recognized. Equally well-known is the difficulty in keeping up to date with the burgeoning multidisciplinary research in this area. Dr. Ulman provides a massive survey of the available literature. The book begins with a section on analytical tools to broaden the understanding of the structure and properties of monolayers and films. Following sections discuss LB films, the preparation and properties of SA monolayers and films, the modeling of LB and SA monolayers, and the application of LB and SA films.
Article
A femtosecond laser based transfection method using off-resonance plasmonic gold nanoparticles is described. For human cancer melanoma cells, the treatment leads to a very high perforation rate of 70%, transfection efficiency three times higher than for conventional lipofection, and very low toxicity (<1%). Off-resonance laser excitation inhibited the fracture of the nanoparticles into possibly toxic DNA intercalating particles. This efficient and low toxicity method is a promising alternative to viral transfection for skin cancer treatment.
Article
Piezoelectric microelectromechanical systems (MEMS) resonant sensors, known for their excellent mass resolution, have been studied for many applications, including DNA hybridization, protein-ligand interactions, and immunosensor development. They have also been explored for detecting antigens, organic gas, toxic ions, and explosives. Most piezoelectric MEMS resonant sensors are acoustic sensors (with specific coating layers) that enable selective and label-free detection of biological events in real time. These label-free technologies have recently garnered significant attention for their sensitive and quantitative multi-parameter analysis of biological systems. Since piezoelectric MEMS resonant sensors do more than transform analyte mass or thickness into an electrical signal (e.g., frequency and impedance), special attention must be paid to their potential beyond microweighing, such as measuring elastic and viscous properties, and several types of sensors currently under development operate at different resonant modes (i.e., thickness extensional mode, thickness shear mode, lateral extensional mode, flexural mode, etc.). In this review, we provide an overview of recent developments in micromachined resonant sensors and activities relating to biochemical interfaces for acoustic sensors.
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Abtract: This work investigates the effect of ultrasound exposure parameters on the sonoporation of KHT-C cells in suspension by perflutren microbubbles. Variations in insonating acoustic pressure (0.05 to 3.5 MPa), pulse frequency (0.5 to 5.0 MHz), pulse repetition frequency (10 to 3000 Hz), pulse duration (4 to 32 μs) and insonation time (0.1 to 900 s) were studied. The number of cells permeabilised to a fluorescent tracer molecule (70 kDa FITC-dextran) and the number of viable cells were measured using flow cytometry. The effect of exposure on the microbubble population was measured using a Coulter counter. Cell viability and membrane permeability were found to depend strongly on the acoustic exposure conditions. Cell permeability increased and viability decreased with increasing peak negative pressure, pulse repetition frequency, pulse duration and insonation time and with decreasing pulse centre frequency. The highest therapeutic ratio (defined as the ratio of permeabilised to nonviable cells) achieved was 8.8 with 32 ± 4% permeabilisation and 96 ± 1% viability at 570 kPa peak negative pressure, 8 μs pulse duration, 3 kHz pulse repetition frequency, 500 kHz centre frequency and 12 s insonation time with microbubbles at 3.3% volume concentration. These settings correspond to an acoustic energy density (ESPPA) of 3.12 J/cm2. Cell permeability and viability did not correlate with bubble disruption. The results indicate that ultrasound exposure parameters can be optimized for therapeutic sonoporation and that bubble disruption is a necessary but insufficient indicator of ultrasound-induced permeabilisation. (E-mail: [email protected] /* */).
Article
E. coli can be transformed to extremely high efficiencies by subjecting a mixture of cells and DNA to brief but intense electrical fields of exponential decay waveform (electroporation). We have obtained 109 to 1010 transformants/μg with strains LE392 and DH5∝, and plasmids pUC18 and pBR329. The process is highly dependent on two characteristics of the electrical pulse: the electric field strength and the pulse length (RC time constant). The frequency of transformation is a linear function of the DNA concentration over at least six orders of magnitude; and the efficiency of transformation is a function of the cell concentration. Most of the surviving cells are competent with up to 80% transformed at high DNA concentration. The mechanism does not appear to include binding of the DNA to the cells prior to entry. Possible mechanisms are discussed and a simple procedure for the practical use of this technique is presented.
Article
A quantitative fluorescent microscopy system was developed to characterize, in real time, the effects of supraphysiological temperatures between 37 degrees and 70 degrees C on the plasma membrane of mouse 3T3 fibroblasts and isolated rat skeletal muscle cells. Membrane permeability was assessed by monitoring the leakage as a function of time of the fluorescent membrane integrity probe calcein. The kinetics of dye leakage increased with increasing temperature in both the 3T3 fibroblasts and the skeletal muscle cells. Analytical solutions derived from a two-compartment transport model showed that, for both cell types, a time-dependent permeability assumption provided a statistically better fit of the model predictions to the data than a constant permeability assumption. This finding suggests that the plasma membrane integrity is continuously being compromised while cells are subjected to supraphysiological temperatures.
Article
We have developed and characterized cellular optoporation with visible wavelengths of light using standard uncoated glass cover slips as the absorptive media. A frequency-doubled Nd:YAG laser pulse was focused at the interface of the glass surface and aqueous buffer, creating a stress wave and transiently permeabilizing nearby cells. Following optoporation of adherent cells, three spatial zones were present which were distinguished by the viability of the cells and the loading efficiency (or number of extracellular molecules loaded). The loading efficiency also depended on the concentration of the extracellular molecules and the molecular weight of the molecules. In the zone farthest from the laser beam (> 60 microns under these conditions), nearly all cells were both successfully loaded and viable. To illustrate the wider applicability of this optoporation method, cells were loaded with a substrate for protein kinase C and the cellular contents then analyzed by capillary electrophoresis. In contrast to peptides loaded by microinjection, optoporated peptide showed little proteolytic degradation, suggesting that the cells were minimally perturbed. Also demonstrating the potential for future work, cells were optoporated and loaded with a fluorophore in the enclosed channels of microfluidic devices.
Article
A streak camera with high spatial and temporal resolution was used for imaging the dynamics of the violent collapse in single-bubble sonoluminescence. The high pressure in the last phase of the bubble collapse leads to the emission of a shock wave, which is launched with a shock velocity of almost 4000 m/s. The shock amplitude decays much faster than approximately 1/r. From the strongly nonlinear propagation the pressure in the vicinity of the bubble can be calculated to be in the range of 40-60 kbar.
Article
Sensory systems use a variety of membrane-bound receptors, including responsive ion channels, to discriminate between a multitude of stimuli. Here we describe how engineered membrane pores can be used to make rapid and sensitive biosensors with potential applications that range from the detection of biological warfare agents to pharmaceutical screening. Notably, use of the engineered pores in stochastic sensing, a single-molecule detection technology, reveals the identity of an analyte as well as its concentration.
Article
Ultrasound, which is routinely used for diagnostic imaging applications, is now being adopted in various drug delivery and other therapeutic applications. Ultrasound has been shown to facilitate the delivery of drugs across the skin, promote gene therapy to targeted tissues, deliver chemotherapeutic drugs into tumours and deliver thrombolytic drugs into blood clots. In addition, ultrasound has also been shown to facilitate the healing of wounds and bone fractures. This article reviews the principles and current status of ultrasound-based treatments.
Article
Sonoporation, in the presence of ultrasound contrast agents (UCA), is a technique that permits the transfer of drugs, including genes, into cells. In this study, the size of the pores created by ultrasound application, and the duration of pore opening, have been characterized via indirect molecular probing and microscopic observation. Internalization of molecules with diameters up to 37 nm was efficient and generally well-tolerated; on the other hand, confocal microscopy revealed that 75 nm particles entered only a few cells when sonoporation was applied. In general, the larger the species to internalize, the poorer the transfer. Direct visualization of pores following insonification, using scanning electron microscopy, was hampered by the presence of numerous villi on the surface of the cells employed (MAT B III), and by the short duration of pore opening. Clearer observations of porated regions were possible using red blood cells. This research (i) confirms that sonoporation is a means with which to achieve macromolecule delivery into cells, and (ii) characterizes in some detail the phenomenon of ultrasound induction of transient pores in the cell membrane.
Article
The structure of a planar supported lipid bilayer (PSLB) prepared by the Langmuir-Blodgett (LB)/Langmuir-Schaefer (LS) method was investigated by sum-frequency vibrational spectroscopy (SFVS). By using asymmetric lipid bilayers composed of selectively deuterated 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) lipids, the orientation of the fatty acid chains and phosphocholine headgroups has been determined independently for both leaflets of the bilayer. The alkyl chains of the lipids were found to be orientated approximately 13 degrees +/- 4 degrees from the surface normal for both leaflets. The lipid chains in both leaflets also contain some gauche content, which is consistent with previous NMR and FTIR studies of similar lipid systems. More importantly, the relative number of gauche defects does not seem to be influenced by the deposition method, LB versus LS. The headgroup orientation for the lipid film in contact with the silica support was determined to be 69 degrees +/- 3 degrees , whereas that in contact with the aqueous phase was 66 degrees +/- 4 degrees from the surface normal. The SFVS results indicate that the structure of the DSPC lipid film in contact with the solid support and the film adjacent to the aqueous phase are nearly identical in structure. These results suggesting the LB/LS deposition method do indeed produce symmetric lipid bilayers. These studies further add to the growing information on the efficacy of PSLBs as suitable models for biological membrane studies.
Article
Ultrasound contrast microbubbles have the ability to enhance endothelial cell permeability and thus may be used as a new way to deliver drugs. It facilitates the transfer of extracellular molecules into cells activated through ultrasound driven microbubbles. The present study is designed to correlate the relationship between microbubble induced cell deformation and enhanced cell membrane permeability. Propidium iodide (PI) was used as a membrane integrity probe. Using high-speed imaging of vibrating microbubbles against endothelial cells and imaging transport of PI into these cells showed a direct correlation between cell deformation and resulting cell membrane permeability. The membrane permeabilization lasted for a short period without affecting endothelial cells viability. We identified that microbubbles are crucial to enhance transient cell membrane permeability. Thus, permeability of individual cells is increased. The roles of ultrasound contrast microbubbles as the trigger for improved drug efficacy are discussed.
Article
Ultrasonic biophysics is the study of mechanisms responsible for how ultrasound and biological materials interact. Ultrasound-induced bioeffect or risk studies focus on issues related to the effects of ultrasound on biological materials. On the other hand, when biological materials affect the ultrasonic wave, this can be viewed as the basis for diagnostic ultrasound. Thus, an understanding of the interaction of ultrasound with tissue provides the scientific basis for image production and risk assessment. Relative to the bioeffect or risk studies, that is, the biophysical mechanisms by which ultrasound affects biological materials, ultrasound-induced bioeffects are generally separated into thermal and non-thermal mechanisms. Ultrasonic dosimetry is concerned with the quantitative determination of ultrasonic energy interaction with biological materials.
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This review offers a critical analysis of the state of the art of medical microbubbles and their application in therapeutic delivery and monitoring. When driven by an ultrasonic pulse, these small gas bubbles oscillate with a wall velocity on the order of tens to hundreds of meters per second and can be deflected to a vessel wall or fragmented into particles on the order of nanometers. While single-session molecular imaging of multiple targets is difficult with affinity-based strategies employed in some other imaging modalities, microbubble fragmentation facilitates such studies. Similarly, a focused ultrasound beam can be used to disrupt delivery vehicles and blood vessel walls, offering the opportunity to locally deliver a drug or gene. Clinical translation of these vehicles will require that current challenges be overcome, where these challenges include rapid clearance and low payload. The technology, early successes with drug and gene delivery, and potential clinical applications are reviewed.
Article
Ultrasound causes high-energy chemistry. It does so through the process of acoustic cavitation: the formation, growth and implosive collapse of bubbles in a liquid. During cavitational collapse, intense heating of the bubbles occurs. These localized hot spots have temperatures of roughly 5000°C, pressures of about 500 atmospheres, and lifetimes of a few microseconds. Shock waves from cavitation in liquid-solid slurries produce high-velocity interparticle collisions, the impact of which is sufficient to melt most metals. Applications to chemical reactions exist in both homogeneous liquids and in liquid-solid systems. Of special synthetic use is the ability of ultrasound to create clean, highly reactive surfaces on metals. Ultrasound has also found important uses for initiation or enhancement of catalytic reactions, in both homogeneous and heterogeneous cases.
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This article reviews the basic physics of ultrasound generation, acoustic field, and both inertial and non-inertial acoustic cavitation in the context of localized gene and drug delivery as well as non-linear oscillation of an encapsulated microbubble and its associated microstreaming and radiation force generated by ultrasound. The ultrasound thermal and mechanical bioeffects and relevant safety issues for in vivo applications are also discussed.
Article
Noninvasive, transient, and local image-guided blood-brain barrier disruption (BBBD) has been demonstrated with focused ultrasound exposure in animal models. Most studies have combined low pressure amplitude and low time average acoustic power burst sonications with intravascular injection of pre-formed micro-bubbles to produce BBBD without damage to the neurons. The BBB has been shown to be healed within a few hours after the exposure. The combination of focused ultrasound beams with MR image guidance allows precise anatomical targeting as demonstrated by the delivery of several marker molecules in different animal models. This method may in the future have a significant impact on the diagnosis and treatment of central nervous system (CNS) disorders. Most notably, the delivery of the chemotherapy agents (liposomal Doxorubicin and Herceptin) has been shown in a rat model.
Article
Ultrasound contrast agents, in the form of gas-filled microbubbles, are becoming popular in perfusion monitoring; they are employed as molecular imaging agents. Microbubbles are manufactured from biocompatible materials, they can be injected intravenously, and some are approved for clinical use. Microbubbles can be destroyed by ultrasound irradiation. This destruction phenomenon can be applied to targeted drug delivery and enhancement of drug action. The ultrasonic field can be focused at the target tissues and organs; thus, selectivity of the treatment can be improved, reducing undesirable side effects. Microbubbles enhance ultrasound energy deposition in the tissues and serve as cavitation nuclei, increasing intracellular drug delivery. DNA delivery and successful tissue transfection are observed in the areas of the body where ultrasound is applied after intravascular administration of microbubbles and plasmid DNA. Accelerated blood clot dissolution in the areas of insonation by cooperative action of thrombolytic agents and microbubbles is demonstrated in several clinical trials.