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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. ...
Intracellular delivery enables the efficient drug delivery into various types of cells and has been a long-term studied topics in modern biotechnology. Targeted delivery with improved delivery efficacy requires considerable requirements. This process is a critical step in many cellular-level studies, such as cellular drug therapy, gene editing delivery, and a series of biomedical research applications. The emergence of micro- and nanotechnology has enabled the more accurate and dedicated intracellular delivery, and it is expected to be the next generation of controlled delivery with unprecedented flexibility. This review focuses on several represented micro- and nanoscale physical approaches for cell membrane disruption-based intracellular delivery and discusses the mechanisms, advantages, and challenges of each approach. We believe that the deeper understanding of intracellular delivery at such low dimension would help the research community to develop more powerful delivery technologies for biomedical applications. Keywords: Drug delivery, Physical approaches, Cell membrane disruption, Low dimension
The construction of artificial cells with specific cell-mimicking functions helps to explore complex biological processes and cell functions in natural cell systems and provides an insight into the origins of life. Bottom-up methods are widely used for engineering artificial cells based on vesicles by the in vitro assembly of biomimetic materials. In this review, the design of artificial cells with a specific function is discussed, by considering the selection of synthetic materials and construction technologies. First, a range of biomimetic materials for artificial cells is reviewed, including lipid, polymeric and hybrid lipid/copolymer materials. Biomaterials extracted from natural cells are also covered in this part. Then, the formation of microscale, giant unilamellar vesicles (GUVs) is reviewed based on different technologies, including gentle hydration, electro-formation, phase transfer and microfluidic methods. Subsequently, applications of artificial cells based on single vesicles or vesicle networks are addressed for mimicking cell behaviors and signaling processes. Microreactors for synthetic biology and cell-cell communication are highlighted here as well. Finally, current challenges and future trends for the development and applications of artificial cells are described.
A surge of research in intracellular delivery technologies is underway with the increased innovations in cell‐based therapies and cell reprogramming. Particularly, physical cell membrane permeabilization techniques are highlighted as the leading technologies because of their unique features, including versatility, independence of cargo properties, and high‐throughput delivery that is critical for providing the desired cell quantity for cell‐based therapies. Amongst the physical permeabilization methods, sonoporation holds great promise and demonstrates to deliver a variety of functional cargos, such as biomolecular drugs, proteins, and plasmids, to various cells including cancer, immune, and stem cells. However, traditional bubble‐based sonoporation methods usually require special contrast agents. Bubble‐based sonoporation methods also have high chances of inducing irreversible damage to critical cell components, lowering the cell viability, and reducing the effectiveness of delivered cargos. To overcome these limitations, several novel non‐bubble‐based sonoporation mechanisms are under development. This review will cover both the bubble‐based and non‐bubble‐based sonoporation mechanisms being employed for intracellular delivery, the technologies being investigated to overcome the limitations of traditional platforms, as well as perspectives on the future sonoporation mechanisms, technologies, and applications. This review presents a comprehensive evaluation of the current state of sonoporation research and its advantages and limitations. Particularly, this review covers the current bubble‐based sonoporation mechanisms and the novel upcoming non‐bubble‐based sonoporation mechanisms and their respective technologies that are utilized to enhance intracellular delivery. This review concludes with a perspective on how the field of sonoporation can advance.
A proposed microsystem system potentially provides accurate and calibration-free measurements based on a combination of front-end particulate matter (PM) separation and back-end PM detection.
Silicon dioxide particles are used to verify the performance of the system’s virtual impactor, and an accurate cutoff diameter of 2 μm in low air flow rates can be obtained.
Experimental results suggest that the proposed microsystem has potential for accurate real-time analysis of PM pollution under complex conditions.
The delicate compositions, structures, and functions of natural cell membranes provide ideal prototypes for the development of functional biomedical polymers. First, the cell membranes, e.g., the red blood cell membrane, can be directly utilized to coat the functional polymeric materials, achieving an excellent biocompatibility and long-term circulation in blood. Second, the lipids (particularly the phospholipids) and glycosylated molecules on the cell membranes help the design of anti-fouling and biocompatible biomaterials, which may also have high afﬁnities to some speciﬁc molecules after a secondary modification. An interesting choline phosphate with the reverse order of phosphate choline in the cell membrane will be specifically discussed here. Third, the transmembrane/peripheral proteins and the channel structure of the cell membrane can also be used as prototypes of biomaterials with various functions, such as the recognition, separation, immobilization, and capture of biomolecules. This review describes such cell membrane bioinspired functional polymers for a variety of biomedical applications including drug/gene delivery, tissue engineering, implant materials, and molecular recognition and diagnosis. The structure-function relationship of these polymeric materials are discussed in detail. Finally, the challenges associated with these cell membrane bioinspired polymeric biomaterials are summarized.
Cellular analysis is a central concept for both biology and medicine. Over the past two decades, acoustofluidic technologies, which marry acoustic waves with microfluidics, have significantly contributed to the development of innovative approaches for cellular analysis. Acoustofluidic technologies enable precise manipulations of cells and the fluids that confine them, and these capabilities have been utilized in many cell analysis applications. In this review article, we examine various applications where acoustofluidic methods have been implemented, including cell imaging, cell mechanotyping, circulating tumor cell phenotyping, sample preparation in clinics, and investigation of cell-cell interactions and cell-environment responses. We also provide our perspectives on the technological advantages, limitations, and potential future directions for this innovative field of methods.
The intracellular delivery efficiency of drug-loaded nanocarriers is often limited by biological barriers arising from the plasma membrane and the cell interior. In this work, the entering of doxorubicin (Dox)-loaded mesoporous silica nanoparticles (MSNs) into cytoplasm was acoustically enhanced through direct penetration with the assistance of hypersound of gigahertz (GHz) frequency. Both fluorescence and cell viability measurements revealed that the therapeutic efficacy of Dox-loaded MSNs were significantly improved by tuning the power and duration of hypersound on demand with a nanoelectromechanical (NEMS) resonator. Mechanism studies with inhibitors illustrated that the membrane defects induced by the hypersound-triggered GHz acoustic streaming facilitated the Dox-loaded MSNs of 100-200 nm to directly penetrate through the cell membrane instead of via the traditional endocytosis, which highly increased the delivery efficiency by avoiding the formation of endosomes. This acoustic method enables the drug carriers to overcome biological barriers of the cell membrane and the endosomes without the limitation of carrier materials, which provides a versatile way of enhanced drug delivery for biomedical applications.
Controllable exchange of molecules between the interior and the external environment of vesicles is critical in drug delivery and micro/nano‐reactors. While many approaches exist to trigger release from vesicles, controlled loading remains a challenge. Here, we show that gigahertz acoustic streaming generated by a nanoelectromechanical resonator can control the loading and release of cargo into/from vesicles. Polymer‐shelled vesicles showed loading and release of molecules both in solution and on a solid substrate. We observed deformation of individual giant unilamellar vesicles and propose that the shear stress generated by gigahertz acoustic streaming induces the formation of transient nanopores in the vesicle membranes. The size of these pores was estimated to be on the order of 100 nm by loading nanoparticles of different sizes into the vesicles. Forming such pores with gigahertz acoustic streaming provides a non‐invasive method to control materials exchange across membranes of different types of vesicles. This method could allow site‐specific release of therapeutics and controlled loading into cells, as well as tunable microreactors.
In this study, stable nano-sized bubbles (nanobubbles [NBs]) were produced using the mechanical agitation method in the presence of perfluorocarbon gases. NBs made with perfluoropropane had a smaller size (around 400 nm) compared to that of those made with perfluorobutane or nitrogen gas. The lipid concentration in NBs affected both their initial size and post-formulation stability. NBs formed with a final lipid concentration of 0.5 mg/ml tended to be more stable, having a uniform size distribution for 24 h at room temperature and 50 h at 4 °C. In vitro gene expression revealed that NBs/pDNA in combination with ultrasound (US) irradiation had significantly higher transfection efficacy in colon C26 cells. Moreover, for in vivo gene transfection in mice left limb muscles, there was notable local transfection activity by NBs/pDNA when combined with US irradiation. In addition, the aged NBs kept at room temperature or 4 °C were still functional at enhancing gene transfection in mice. We succeeded in p...
Controlled drug release has a high priority for the development of modern medicine and biochemistry. To develop a versatile method for controlled release, a miniaturized acoustic gigahertz (GHz) resonator is designed and fabricated which can transfer electric supply to mechanical vibrations. By contacting with liquid, the GHz resonator directly excites streaming flows and induces physical shear stress to tear the multilayered polyelectrolyte (PET) thin films. Due to the ultra-high working frequency, the shear stress is greatly intensified, which results in a controlled disassembling of the PET thin films. This technique is demonstrated as an effective method to trigger and control the drug release. Both theory analysis and controlled release experiments prove the thin film destruction and the drug release..
Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.
When high-amplitude, short-duration pulsed electric fields are applied to cells and tissues, the membrane permeability of the cells and tissue is increased. This increase in permeability is currently explained by the temporary appearance of aqueous pores within the cell membrane, a phenomenon termed electroporation. During the past four decades, advances in fundamental and experimental electroporation research have allowed for the translation of electroporation-based technologies to clinic. In this review, we describe the theory and current applications of electroporation in medicine and then discuss current challenges in electroporation research and barriers to a more extensive spread of these clinical applications.
In the past two decades, research has underlined the potential of ultrasound and microbubbles to enhance drug delivery. However, there is less consensus on the biophysical and biological mechanisms leading to this enhanced delivery. Sonoporation, i.e. the formation of temporary pores in the cell membrane, as well as enhanced endocytosis is reported. Because of the variety of ultrasound settings used - and corresponding microbubble behavior, a clear overview is missing. Therefore, in this review, the mechanisms contributing to sonoporation are categorized according to three ultrasound settings: i) low intensity ultrasound leading to stable cavitation of microbubbles, ii) high intensity ultrasound leading to inertial cavitation with microbubble collapse, and iii) ultrasound application in the absence of microbubbles. Using low intensity ultrasound, the endocytotic uptake of several drugs could be stimulated, while short but intense ultrasound pulses can be applied to induce pore formation and the direct cytoplasmic uptake of drugs. Ultrasound intensities may be adapted to create pore sizes correlating with drug size. Small molecules are able to diffuse passively through small pores created by low intensity ultrasound treatment. However, delivery of larger drugs such as nanoparticles and gene complexes, will require higher ultrasound intensities in order to allow direct cytoplasmic entry.
In this paper, we demonstrate a new single-cell optoporation and transfection technique using a femtosecond Gaussian laser beam and optical tweezers. Tightly focused near-infrared (NIR) femtosecond laser pulse was employed to transiently perforate the cellular membrane at a single point in MCF-7 cancer cells. A distinct technique was developed by trapping the microparticle using optical tweezers to focus the femtosecond laser precisely on the cell membrane to puncture it. Subsequently, an external gene was introduced in the cell by trapping and inserting the same plasmid-coated microparticle into the optoporated cell using optical tweezers. Various experimental parameters such as femtosecond laser exposure power, exposure time, puncture hole size, exact focusing of the femtosecond laser on the cell membrane, and cell healing time were closely analyzed to create the optimal conditions for cell viability. Following the insertion of plasmid-coated microparticles in the cell, the targeted cells exhibited green fluorescent protein (GFP) under the fluorescent microscope, hence confirming successful transfection into the cell. This new optoporation and transfection technique maximizes the level of selectivity and control over the targeted cell, and this may be a breakthrough method through which to induce controllable genetic changes in the cell.
This paper describes a study of the electrical properties of supported lipid bilayer membranes on semiconductor and gold surfaces. The study is aimed to foster the understanding of supported membrane systems and to allow the rational design of biosensor assays for ion channel analysis. Impedance spectroscopy was applied for the electrical characterization of the supported membrane systems. A novel equivalent circuit model is introduced for the data evaluation, which accounts for the deviation of the impedance response of supported membranes from that of an ideal RC element. As a result of the improved accordance of model and data, the resistance and the capacity of supported membranes can be determined more accurately and independently from each other. Experimental results describe the phenomenology of the electrical properties of supported bilayers regarding variations in preparation, composition, and environmental conditions. We discuss the findings in terms of membrane−substrate interactions and models of membrane permeability. The important role of the electrostatics between the lipid bilayer and the solid substrate for the formation of an electrically dense supported membrane is identified. Bilayer permeability models explain the correlation between the structure of the lipid bilayer and its insulating properties. These models are also in accordance with the observed dependence of the electrical resistance of the lipid bilayer on the temperature and the ion concentration of the electrolyte.
For some time, FBAR technology has lagged behind ceramic
technology and surface acoustic wave resonator (SAW) technology for
commercial applications. There were several technologies that had to be
developed before FBAR technology became viable for rf filters. First, a
process is needed that can make the resonators manufacturable, robust
and repeatable. Second, maximizing the coupling coefficient, k<sub>t
</sub><sup>2</sup> and the Q of the resonator (k<sub>t</sub><sup>2</sup>
Q product) is necessary. Another technology needed is a method to
eliminate ripple (or "suck out") associated with lateral mode
excitation. Lastly, a method is needed for maintaining a uniform
thickness (for frequency control and a means to target frequency to
within +-0.03%). If one overcomes these sets of hurdles, the rewards are
high. The Quality factor, Q, inherent in these structures is impressive
(over 2500) and the intrinsic k<sub>t</sub><sup>2</sup> has been
inferred to be close to the theoretical maximum of 6.5%. The k<sub>t
</sub><sup>2</sup> Q product (Figure of Merit for FBAR filters) have
been as high as 100 for our devices. These two properties can be
combined in a filter to achieve low pass band insertion loss and
extremely sharp skirts. One intrinsic advantage of FBAR over SAW
technology is the ability to handle input power in excess of 4 Watts.
Resistance to Electrical Static Discharge (ESD) is another desirable
property of FBAR devices. Finally, FBAR technology is intrinsically a
"low temperature" process technology-compatible with semiconductor
technology. This implies future integration of FBARs with semiconductor
The purpose of this study was to develop a unified model capable of explaining the mechanisms of interaction of ultrasound and biological tissue at both the diagnostic nonthermal, noncavitational (<100 mW · cm(-2)) and therapeutic, potentially cavitational (>100 mW · cm(-2)) spatial peak temporal average intensity levels. The cellular-level model (termed "bilayer sonophore") combines the physics of bubble dynamics with cell biomechanics to determine the dynamic behavior of the two lipid bilayer membrane leaflets. The existence of such a unified model could potentially pave the way to a number of controlled ultrasound-assisted applications, including CNS modulation and blood-brain barrier permeabilization. The model predicts that the cellular membrane is intrinsically capable of absorbing mechanical energy from the ultrasound field and transforming it into expansions and contractions of the intramembrane space. It further predicts that the maximum area strain is proportional to the acoustic pressure amplitude and inversely proportional to the square root of the frequency (ε A,max ∝ P(A)(0.8f - 0.5) and is intensified by proximity to free surfaces, the presence of nearby microbubbles in free medium, and the flexibility of the surrounding tissue. Model predictions were experimentally supported using transmission electron microscopy (TEM) of multilayered live-cell goldfish epidermis exposed in vivo to continuous wave (CW) ultrasound at cavitational (1 MHz) and noncavitational (3 MHz) conditions. Our results support the hypothesis that ultrasonically induced bilayer membrane motion, which does not require preexistence of air voids in the tissue, may account for a variety of bioeffects and could elucidate mechanisms of ultrasound interaction with biological tissue that are currently not fully understood.
Ultrasound exposure (USE) in the presence of microbubbles (MCB) (e.g. contrast agents used to enhance ultrasound imaging) increases plasmid transfection efficiency in vitro by several orders of magnitude. Formation of short-lived pores in the plasma membrane ('sonoporation'), up to 100 nm in effective diameter lasting a few seconds, is implicated as the dominant mechanism, associated with acoustic cavitation. Ultrasound enhanced gene transfer (UEGT) has also been successfully achieved in vivo, with reports of spatially restricted and therapeutically relevant levels of transgene expression. Loading MCB with nucleic acids and/or disease-targeting ligands may further improve the efficiency and specificity of UEGT such that clinical testing becomes a realistic prospect.
The majority of infectious diseases enter the body through mucosal membranes that line the ocular, nasal, oral, vaginal and rectal surfaces. As infections can be effectively prevented by instigating a local immune response in the immunocyte-rich regions of the mucosa, an efficacious route of vaccine administration is to directly target their delivery to these surfaces. It is nevertheless challenging to provide sufficient driving force to penetrate both the mucus lining as well as the epithelial barrier of the mucosal surfaces, which are designed to effectively keep foreign entities out, but not excessively such that the therapeutic agent penetrates deeper into the vascularised submucosal regions where they are mostly taken up by the systemic circulation, thus resulting in a far weaker immune response. In this work, we demonstrate the possibility of controllably localising and hence maximising the delivery of both small and large molecule model therapeutic agents in the mucosa of a porcine buccal model using high frequency acoustics. Unlike their low (kHz order) frequency bulk ultrasonic counterpart, these high frequency (>10 MHz) surface waves do not generate cavitation, which leads to large molecular penetration depths beyond the 100 μm order thick mucosal layer, and which has been known to cause considerable cellular/tissue damage and hence scarring. Through system parameters such as the acoustic irradiation frequency, power and exposure duration, we show that it is possible to tune the penetration depth such that over 95% of the delivered drug are localised within the mucosal layer, whilst preserving their structural integrity.
Nonspecific binding (NSB) is a general issue for surface based biosensors. Various approaches have been developed to prevent or remove the NSBs. However, these approaches either increased the background signals of the sensors or limited to specific transducers interface. In this work, we developed a hydrodynamic approach to selectively remove the NSBs using a microfabricated hypersonic resonator with 2.5 gigahertz (GHz) resonant frequency. The high frequency device facilitates to generate multiple controlled micro-vortices which then create cleaning forces at the solid-liquid interfaces. The competitive adhesive and cleaning forces have been investigated using the finite element method (FEM) simulation, identifying the feasibility of the vortices induced NSB removal. NSB proteins have been selectively removed experimentally both on the surface of the resonator and on other substrates which contact the vortices. Thus, the developed hydrodynamic approach is believed to be a simple and versatile tool for NSB removal and compatible to many sensor systems. The unique feature of the hypersonic resonator is that it can be used as a gravimetric sensor as well, thus a combined NSB removal and protein detection dual functional biosensor system is developed.
Ultrasound is widely used for medical diagnosis and increasingly for therapeutic purposes. An understanding of the bio-effects of sonography is important for clinicians and scientists working in the field because permanent damage to biological tissues can occur at high levels of exposure. Here the underlying principles of thermal mechanisms and the physical interactions of ultrasound with biological tissues are reviewed. Adverse health effects derived from cellular studies, animal studies and clinical reports are reviewed to provide insight into the in vitro and in vivo bio-effects of ultrasound.
Efficient delivery of genes and therapeutic agents to the interior of the cell is critical for
modern biotechnology. Herein, a new type of chemical-free cell poration method—
hypersonic poration—is developed to improve the cellular uptake, especially the
nucleus uptake. The hypersound (≈GHz) is generated by a designed piezoelectric
nano-electromechanical resonator, which directly induces normal/shear stress and
“molecular bombardment” effects on the bilayer membranes, and creates reversible
temporal nanopores improving the membrane permeability. Both theory analysis
and cellular uptake experiments of exogenous compounds prove the high delivery
efficiency of hypersonic poration. Since target molecules in cells are accumulated with
the treatment, the delivered amount can be controlled by tuning the treatment time.
Furthermore, owing to the intrinsic miniature of the resonator, localized drug delivery
at a confined spatial location and tunable arrays of the resonators that are compatible
with multiwell plate can be achieved. The hypersonic poration method shows great
delivery efficacy combined with advantage of scalability, tunable throughput, and
simplification in operation and provides a potentially powerful strategy in the field of
molecule delivery, cell transfection, and gene therapy.
The advancement in micro/nanotechnologies has been continuously providing possibilities for inventing novel biochemical sensors. However, variations in the transducer type can cause different sensing results due to the differences in their mechanisms of analyzing biomolecular interactions. In this work, we focused on the comparative analysis of static and non-static assays for molecular interactions using on-chip integrated extended-gate field effect transistor (EGFET) as a static sensing interface and solidly mounted resonator (SMR) as a non-static sensing interface. Analysis of polyelectrolytes (PETs) surface assembly and antigen-antibody interaction using the two types of biochemical sensors presented consistent and complementary sets of information. Meanwhile, due to the difference in their operating mechanisms, variations on the detection efficiency, kinetics and thermodynamics were observed. Our results highlighted the critical dependence of signal detection on biochemical sensors’ operating mechanisms and provided a valuable guidance for static and non-static assays for biomolecular detections.
Ultrasound-triggered drug delivery is now becoming a mature technology with first patients enrolling in clinical
trials. Having a clear overviewof the field is complicated as it mixes ultrasound physics and biological effects, particle formulation, and pharmacokinetics and biodistribution. The scope of this review is to move from basics to the latest developments of combined techniques using ultrasound for triggering drug release. Generalities on ultrasound are first given to better understand the parameters on which the clinician can operate to modulate the amount of delivered energy. Ultrasound effects on biological tissues such as thermal effects, mechanical effects and radiation forces are also presented. The second part of this review deals with the combination of ultrasound and drug delivery systems to enhance the efficacy of current cancer treatment. The in vivo behavior of drug delivery systems and howultrasounds can be combined to improve treatment efficacy are detailed. The example of ThermoDox®, a new formulation of thermosensitive liposomes undergoing a phase III clinical trial, is particularly discussed on the basis of the available clinical data. Through the present article, researchers will be able to better grasp the different levels of complexity when designing an efficient formulation to be combinedwith ultrasound.
Ultrasound contrast agents are valuable in diagnostic ultrasound imaging, and they increasingly show potential for drug delivery. This review focuses on the acoustic behavior of flexible-coated microbubbles and rigid-coated microcapsules and their contribution to enhanced drug delivery. Phenomena relevant to drug delivery, such as non-spherical oscillations, shear stress, microstreaming, and jetting will be reviewed from both a theoretical and experimental perspective. Further, the two systems for drug delivery, co-administration and the microbubble as drug carrier system, are reviewed in relation to the microbubble behavior. Finally, future prospects are discussed that need to be addressed for ultrasound contrast agents to move from a pre-clinical tool into a clinical setting.
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
A potentiometric non-enzymatic sensor using off-chip extended-gate field effect transistor (EGFET) with a ferrocenyl-alkanethiol modified gold electrode is demonstrated for determining the uric acid concentration in human serum and urine. Hexacyanoferrate (II) and (III) ions are used as redox reagent. This potentiometric sensor measures the interface potential on the ferrocene immobilized gold electrode, which is modulated by the redox reaction between uric acid and hexacyanoferrate ions. The device shows a near Nernstian response to uric acid and is highly specific. The interference that comes from glucose, bilirubin, ascorbic acid and hemoglobin is negligible in normal concentration range of these interferents. The sensor also exhibits excellent long term reliability. This extended gate field effect transistor based sensors can be used as a point of care UA testing tool, due to the small size, low cost, and low sample volume consumption.
Trehalose was introduced into suspended primary rat hepatocytes through pathways resulting from thermally induced alterations of the cellular membrane. The hepatocytes were suspended in a diluted hepatocyte culture medium (medium:dH2O = 1:2) with 0.4 M trehalose during thermal treatments. A significant amount of cytoplasmic trehalose (0.07 M) was detected using high-performance liquid chromatography (HPLC) after heating hepatocytes to 39°C for 10 min in trehalose-supplemented medium. High cell viability (approximately 90%) was retained. The cytoplasmic trehalose concentration reached a plateau (approximately 0.16 M) after heating for 1-2 h. However, the cell viability decreased significantly after 30 min of heating (< approximately 72%). It was further found that by repetitive heating between 0°C and 39°C every 10 min for 1 h (0-39°C, 1 h), high cell viability (approximately 83%) could be maintained and a high cytoplasmic trehalose concentration (approximately 0.13 M) could be obtained. The trehalose-laden hepatocytes (0-39°C, 1 h) were cultured in a double-collagen gel sandwich system for 15 days. They retained normal morphology and produced a normal distribution of F-actin filaments. Furthermore, the hepatospecific functions of urea production and albumin synthesis were similar to those of control hepatocytes kept in fresh medium on ice for one hour. In short, trehalose can be introduced effectively into primary rat hepatocytes by challenging the cells with super-zero to mild hyperthermic (39°C) temperatures. Future studies will focus on the development of effective protocols for both cryopreservation and lyopreservation of trehalose-laden hepatocytes.
A method for the generation and detection of hypersonic waves, which has
only been briefly described earlier, together with some absorption
measurements in quartz, is discussed in some detail. Further
measurements of the hypersonic absorption in quartz at different crystal
orientations and after neutron irradiation are reported. The results are
in qualitative agreement with a phonon-phonon relaxation process.
Unlike the quartz crystal microbalance, which has been used extensively for the analysis of biochemical interactions, only few measurements with biochemical adsorbent have been done with film bulk acoustic resonators (FBAR). In this paper, the FBAR behaviour on exposure to a lipid vesicle solution and the formation of a polyelectrolyte multilayer structure is investigated and compared with the results obtained with the quartz crystal microbalance. Differences in the resonator response were found between the two techniques and depending on the resonators resonance frequency ranging from the MHz to the GHz regime. As an explanation, we suggest that the penetration depth and the influence on viscoelastic properties, which are both known to be frequency dependent, cause the variations in the results. As a consequence, the higher operating resonance frequencies of the FBAR increase the sensitivity to changes in the viscoelasticity of the adsorbent and also decrease the sensing length of the device.
The interaction between ultrasound pulses and microbubbles is known to generate acoustic cavitation that may puncture biological cells. This work presents new experimental findings on the bioeffects of ultrasound-microbubble mediated cavitation in plant cells with emphasis on direct observations of morphological impact and analysis of viability trends in tobacco BY-2 cells that are widely studied in higher plant physiology. The tobacco cell suspensions were exposed to 1 MHz ultrasound pulses in the presence of 1% v/v microbubbles (10% duty cycle; 1 kHz pulse repetition frequency; 70 mm between probe and cells; 1-min exposure time). Few bioeffects were observed at low peak negative pressures (<0.4 MPa) where stable cavitation presumably occurred. In contrast, at 0.9 MPa peak negative pressure (with more inertial cavitation activities according to our passive cavitation detection results), random pores were found on tobacco cell wall (observed via scanning electron microscopy) and enhanced exogenous uptake into the cytoplasm was evident (noted in our fluorescein isothiocyanate dextran uptake analysis). Also, instant lysis was observed in 23.4% of cells (found using trypan blue staining) and programmed cell death was seen in 23.3% of population after 12 h (determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling [TUNEL]). These bioeffects generally correspond in trend with those for mammalian cells. This raises the possibility of developing ultrasound-microbubble mediated cavitation into a targeted gene transfection paradigm for plant cells and, conversely, adopting plant cells as experimental test-beds for sonoporation-based gene therapy in mammalian cells.
Transdermal drug delivery has many potential advantages, but the skin's poorly-permeable stratum corneum blocks delivery of most drugs at therapeutic levels. Short high-voltage pulses have been used to electroporate the skin's lipid bilayer barriers and thereby deliver compounds at rates increased by as much as four orders of magnitude. Evidence that the observed flux enhancement is due to physical alteration of the skin by electroporation, as opposed to only providing an iontophoretic driving force, is supported by a number of different transport, electrical and microscopy studies. Practical applications of electroporation's unique effects on skin are motivated by large flux increases for many different compounds, rapidly responsive delivery profiles, and efficient use of skin area and electrical charge. Greater enhancement can be achieved by combining skin electroporation with iontophoresis, ultrasound, and macromolecules. Sensation due to electroporation can be avoided by using appropriate electrical protocols and electrode design. To develop skin electroporation as a successful transdermal drug delivery technology, the strong set of existing in vitro mechanistic studies must be supplemented with studies addressing in vivo/clinical issues and device design.
We report here that a hairpin-structured DNA that possesses an anti-ATP aptamer sequence successfully detected target ATP or adenosine in a temperature-dependent manner by nanoscale intramolecular displacement on the surface of a gold electrode as an extended gate of a field-effect transistor (FET). The structural switching of the hairpin aptamer from closed loop to open-loop conformations was accompanied by the release of the preloaded DNA binder (DAPI) from the stem part of the hairpin aptamer into the solution phase. The loss of intrinsic positive charges of DAPI (2+) from the diffusion layer at the gate/solution nano-interface as a result of target capturing was responsible for generating a specific signal by the field-effect. We emphasize a new aspect of the structured DNA aptamer in combination with FET: the DAPI-loaded hairpin aptamer successfully detected even uncharged adenosine, which remains a major challenge for FET-based biosensors. Given the simplicity in design of the primary and secondary structures of oligonucleotide aptamers, it is easy to apply this technology to a wide variety of bio-analytes, irrespective of their electric charges. In view of these advantages, our findings may offer a new trend in the design of stimuli-responsive "smart" biomolecular switches for semiconductor-based biosensors.
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.
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.
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.
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]
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.