Article

Hypersonic Poration: A New Versatile Cell Poration Method to Enhance Cellular Uptake Using a Piezoelectric Nano-Electromechanical Device

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Abstract

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.

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... 107 Hypersound, defined as ultrasound with frequency N 1 GHz and generated by bulk acoustic wave resonator, has been recently reported for drug delivery applications. 108 In this type of device, thin-film piezoelectric material is sandwiched between two metal electrodes to achieve a high resonate frequency. Such structure also guarantees the device stability at high power input (up to a few watts). ...
... Zhang et al. 108 used the GHz resonator to develop a novel cell poration method. Hypersound was used to stimulate cells and induce the transient nanopores to achieve efficient delivery of exogenous molecules. ...
... (a) Schematic of cavitation 101 ; (b) schematic of the acoustic exposure apparatus used to investigate the intracellular delivery of fluorescent marker and cytoskeleton dynamics induced by sonoporation 97 ; (c) side (top) and perspective (bottom) view schematics of the experimental setup 106 ; (d) acoustic transfection of adherent cells 107 ; (e) schematic of the integrated sensing system 111 ; (f) hypersonic wave generated by a nanoelectromechanical resonator.108 ...
Article
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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
... Such resonators have recently been reported by us to generate high-speed (> m s À1 ) acoustic streaming with strong forces (> nn), which has been applied to enhance the solution mixing in microfluidic chips [39] and to remove nonspecific binding at solid-liquid interfaces. [40] Since acoustic streaming can exert mechanical forces on cells that are immobilized at the solid-liquid interface, [41] we envisaged that vesicles, which are soft and hollow structures, would also be affected and could experience ...
... Such resonators have recently been reported by us to generate high-speed (> m s À1 ) acoustic streaming with strong forces (> nn), which has been applied to enhance the solution mixing in microfluidic chips [39] and to remove nonspecific binding at solid-liquid interfaces. [40] Since acoustic streaming can exert mechanical forces on cells that are immobilized at the solid-liquid interface, [41] we envisaged that vesicles, which are soft and hollow structures, would also be affected and could experience mechanical deformation under such acoustic stimulation. We hypothesized that owing to the fluidic nature of the lipid membranes, mechanical deformation of the vesicles might induce transient pores in the membrane, which would change the membrane permeability and facilitate materials exchange between the interior and exterior of the vesicles (Scheme 1). ...
Article
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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.
... It has recently been confirmed that the strong acoustic force driven by BAW can realize in-situ mixing and microparticles concentrating [36,37]. In addition, recent works from our group also found that this strong acoustic streaming could drive the membrane of mammalian cells [38] as well as inducing deformation [35] and differentiation of cells [39], to facilitate the biological applications. ...
... But it always needs a long incubation time from several hours to days [13,[43][44][45]. Our previous results have reported that the shear stress generated by the acoustic streaming could act on the membrane of cells and produce temporary pore on the membrane [38], that facilitates the foreign materials cross the cell plasma membrane and directly enter the cytoplasm (Fig. 3a). As shown in Fig. 3b, when incubating the CDs with HeLa cells directly, no obvious green fluorescence of CDs appeared, suggesting that few CDs entered cells. ...
Article
Currently, researches on nanomaterials have been restricted by slow and multistep synthesis procedures. Herein, we demonstrate an ultrafast, one step method of purification and delivery of quantum dots into living cells, actuated by the acoustic streaming (AS) produced through a gigahertz resonator. Results demonstrate that the impurities in the carbon dots (CDs) can be extracted immediately aided by the acoustic forcing, with extra high purification efficiency of 93%. The system can also efficiently deliver the CDs into cells, showing excellent nucleus and mitochondria uptake under 3 min of acoustic streaming treatment, and making the organelles of cells to be recorded more easily and simultaneously. More importantly, the AS is found to further accelerate the bioreaction inside the cells, thus realizes the enhanced biosensing of Fe³⁺ in single living cells. This work develops a novel type of multifunctional method for effective purification, intracellular delivery and biosensing of nanomaterials, inspiring the biological/medical nanotechnology researches at subcellular level.
... Such resonators have recently been reported by us to generate high-speed (> m s À1 ) acoustic streaming with strong forces (> nn), which has been applied to enhance the solution mixing in microfluidic chips [39] and to remove nonspecific binding at solid-liquid interfaces. [40] Since acoustic streaming can exert mechanical forces on cells that are immobilized at the solid-liquid interface, [41] we envisaged that vesicles, which are soft and hollow structures, would also be affected and could experience ...
... Such resonators have recently been reported by us to generate high-speed (> m s À1 ) acoustic streaming with strong forces (> nn), which has been applied to enhance the solution mixing in microfluidic chips [39] and to remove nonspecific binding at solid-liquid interfaces. [40] Since acoustic streaming can exert mechanical forces on cells that are immobilized at the solid-liquid interface, [41] we envisaged that vesicles, which are soft and hollow structures, would also be affected and could experience mechanical deformation under such acoustic stimulation. We hypothesized that owing to the fluidic nature of the lipid membranes, mechanical deformation of the vesicles might induce transient pores in the membrane, which would change the membrane permeability and facilitate materials exchange between the interior and exterior of the vesicles (Scheme 1). ...
Article
Full-text available
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.
... 36 It has been reported that hypersound can be applied to enhance the delivery of drug molecules into cancer cells by creating transient nanopores in the cell membrane, which showed no cytotoxicity. 37 The mechanical stress on the membrane surface is significantly enhanced by hypersonic poration compared with the conventional ultrasonic treatment. Furthermore, hypersound has also been applied in a layer-bylayer (LbL) system to control the disassembly of supramolecular membrane structures. ...
... The polygonal shape of the NEMS resonator was designed to enhance its main-mode vibration while minimizing the parasitic effect. 37 By coupling the vertical electric field through a specific piezoelectric coefficient, the resonator vibrates in a longitudinal mode and generates hypersound of gigahertz (GHz) frequency. 47 A quarter-wavelength Bragg reflector was placed under the resonance structure to avoid the dissipation of energy into the silicon substrate. ...
Article
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.
... On the other hand, the high frequencies and high P max+ of PA waves lead to high stress gradients and high impulses that enable less common mechanisms of interaction between US and cells. GHz frequency acoustic waves have been shown to interact with cell membranes and contribute to membrane deformation 39 . Reversible poration of the cell membrane with hypersound was shown to transfect plasmid DNA encoding GFP to HeLa cells 39 . ...
... GHz frequency acoustic waves have been shown to interact with cell membranes and contribute to membrane deformation 39 . Reversible poration of the cell membrane with hypersound was shown to transfect plasmid DNA encoding GFP to HeLa cells 39 . A limitation of piezoelectric generation of such hypersound is the small size of the piezoelectric resonator. ...
Article
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Photoacoustic transfection consists in the use of photoacoustic waves, generated in the thermoelastic expansion of a confined material absorbing a short pulse of a laser, to produce temporary mechanical deformations of the cell membrane and facilitate the delivery of plasmid DNA into cells. We show that high stress gradients, produced when picosecond laser pulses with a fluence of 100 mJ/cm2 are absorbed by piezophotonic materials, enable transfection of a plasmid DNA encoding Green Fluorescent Protein (gWizGFP, 3.74 MDa) in COS-7 monkey fibroblast cells with an efficiency of 5% at 20 °C, in 10 minutes. We did not observe significant cytotoxicity under these conditions. Photoacoustic transfection is scalable, affordable, enables nuclear localization and the dosage is easily controlled by the laser parameters.
... Previously, our group used an SMR for driving biomolecules in fluid and found that the dynamic behavior of the particles was more intense than expected [20]. It was guessed that the phenomenon may be related to the fluid working environment. ...
Article
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The very small vibration of a solidly-mounted resonator (SMR) in fluid may trigger a relatively large motion of the covering fluid, which was implied by our protein-related experimental results. Therefore, a series of experimental methods for characterizing the mechanical longitudinal vibration of the SMR and the corresponding out-of-plane dynamic response of the fluid above the SMR surface is described in this paper. A SMR device with theoretical resonance frequency of 2.5 GHz was driven by an amplitude-modulated (AM) signal, in which the amplitude is modulated by a signal of the second resonance frequency of the atomic force microscope (AFM) cantilever. A lock-in amplifier is used to demodulate the vibration response of the AFM cantilever, which is proportional to the amplitude of the sample vibration in contact mode and tapping mode. The amplitude-frequency curve of the SMR surface is obtained in contact mode with a relatively stronger interaction force between the AFM tip and the SMR surface. The amplitude-frequency curve of the motion of the liquid above the SMR device and the peak amplitude of the fluid at different distances above the SMR surface are measured in tapping mode with a relatively weak interaction force between the AFM tip and the fluid sample.
... In yet another study, a much higher frequency at the hypersound level induced cell membrane perforation. A nanoelectromechanical resonator was developed to generate an acoustic wave at 1.6 GHz, creating membrane pores of around 200 nm [120]. ...
Article
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Gene editing is a versatile technique in biomedicine that promotes fundamental research as well as clinical therapy. The development of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) as a genome editing machinery has accelerated the application of gene editing. However, the delivery of CRISPR components often suffers when using conventional transfection methods, such as viral transduction and chemical vectors, due to limited packaging size and inefficiency toward certain cell types. In this review, we discuss physical transfection methods for CRISPR gene editing which can overcome these limitations. We outline different types of physical transfection methods, highlight novel techniques to deliver CRISPR components, and emphasize the role of micro and nanotechnology to improve transfection performance. We present our perspectives on the limitations of current technology and provide insights on the future developments of physical transfection methods.
... Electroporation is most widely accepted with demonstrated efficacy of DNA 23,24 , RNA 25,26 and even protein delivery 27 ; however, this method can produce unacceptable levels of cell death, DNA damage and electric field-induced agglomeration of certain nanomaterials 8 . While electroporation and sonoporation are relatively mature technologies, the last decade has witnessed the emergence of several alternative injury/diffusion-based delivery methods including optoporation 28 , thermoporation 29 , high-frequency acoustic transfection 30 , hypersonic poration 31 , and continuous-flow, shear-based mechanoporation [32][33][34][35] . These technologies are often amenable to miniaturization, enabling rapid advancement of intracellular delivery applications through introduction of microfluidics and nanotechnology 2,3 . ...
Article
Full-text available
Delivery of large and structurally complex target molecules into cells is vital to the emerging areas of cellular modification and molecular therapy. Inadequacy of prevailing in vivo (viral) and in vitro (liposomal) gene transfer methods for delivery of proteins and a growing diversity of synthetic nanomaterials has encouraged development of alternative physical approaches. Efficacy of injury/diffusion-based delivery via shear mechanoporation is largely insensitive to cell type and target molecule; however, enhanced flexibility is typically accompanied by reduced gene transfer effectiveness. We detail a method to improve transfection efficiency through coordinated mechanical disruption of the cell membrane and electrophoretic insertion of DNA to the cell interior. An array of micromachined nozzles focuses ultrasonic pressure waves, creating a high-shear environment that promotes transient pore formation in membranes of transmitted cells. Acoustic Shear Poration (ASP) allows passive cytoplasmic delivery of small to large nongene macromolecules into established and primary cells at greater than 75% efficiency. Addition of an electrophoretic action enables active transport of target DNA molecules to substantially augment transfection efficiency of passive mechanoporation/diffusive delivery without affecting viability. This two-stage poration/insertion method preserves the compelling flexibility of shear-based delivery, yet substantially enhances capabilities for active transport and transfection of plasmid DNA.
... Piezoelectric transducer (PZT) has also been incorporated into microfluidics to generate acoustic streaming and mixing (Madison et al. 2017). Compared with the above two strategies, thin-film-type device fabricated by MEMS technology as another avenue has the advantages of smaller size (~ µm) and higher frequency (~ GHz) (Pang et al. 2012;Zhang et al. 2017), which might contribute to high level of integration for on-chip applications and large acoustic streaming force for higher vortices velocity (~ m/s). However, in the realm of acoustic mixing, the research on GHz acoustic mixing (Cui et al. 2016;Qu et al. 2017;Wang et al. 2017) is still limited. ...
Article
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Rapid and reliable micromixing requires continuous improvement to renovate more powerful microfluidics chip for chemosynthesis, biological assay, and drug purification. In this work, we realized rapid in-situ mixing in droplets on a closed electro-microfluidic chip. Electrowetting and 2.5 GHz acoustic wave streaming were coupled into a monolithic chip for the manipulation and active mixing of microdroplets, respectively. Finite-element analysis simulation provided three-dimensional illustrations of turbulent flow pattern, fluid velocity, and vortices core locations. We carried out mixing experiments on different scales from nanoscale molecules to microscale particles, accelerating mixing efficiency by more than 50 times compared with pure diffusion. In the enzyme catalytic reaction experiment for biological assay demonstration, mixing efficiency of biological samples improves by about one order of magnitude compared with conventional 96-well-plate assay. Limited temperature rising of mixing in microdroplets validates biological safety, which guarantees potentials of the chip in various biochemical analyses and medical applications.
... 17 Recently, we demonstrated the use of a GHz-acoustic-wave resonator to generate a localized micro-vortex, which we applied to some exciting applications including microfluidic mixing, 18 acoustofluidic tweezering, 19 and cellar surgery. 20 However, at present, almost all of the developed high frequency acoustofluidic techniques are at their very earliest developmental stages, and their theoretical principles have yet to be established. In this work, we investigate and reveal the properties of GHz-acoustic-induced vortex streaming, including GHz acoustic device, streaming generation, and microscale streaming characterizations, and present a full picture of GHz acoustic streaming and its microfluidic applications. ...
Article
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a r t i c l e i n f o Even as gigahertz (GHz) acoustic streaming has developed into a multi-functional platform technology for biochemical applications, including ultrafast microfluidic mixing, microparticle operations, and cellar or vesicle surgery , its theoretical principles have yet to be established. This is because few studies have been conducted on the use of such high frequency acoustics in microscale fluids. Another difficulty is the lack of velocimetry methods for microscale and nanoscale fluidic streaming. In this work, we focus on the basic aspects of GHz acoustic streaming, including its micro-vortex generation principles, theoretical model, and experimental characterization technologies. We present details of a weak-coupled finite simulation that represents our current understanding of the GHz-acoustic-streaming phenomenon. Both our simulation and experimental results show that the GHz-acoustic-induced interfacial body force plays a determinative role in vortex generation. We carefully studied changes in the formation of GHz acoustic streaming at different acoustic powers and flow rates. In particular, we developed a microfluidic-particle-image velocimetry method that enables the quantification of streaming at the microscale and even nanoscale. This work provides a full map of GHz acoustofluidics and highlights the way to further theoretical study of this topic.
... Owning to the development of microsystem and nanotechnology, acoustic devices based on piezoelectric materials have gained increasing attention in biochemical research field [41][42][43][44] which is due to their low cost, batch manufacturing, small volume and noninvasive to biomolecules [45][46][47]. Here, we demonstrated a novel and versatile controlled release approach using gigahertz ultrasound (hypersound) induced by a nano-electromechanical acoustic resonator composed of ultra-thin material layers (several tens to hundreds of nanometers thick). ...
Article
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Background Controllable and multiple DNA release is critical in modern gene-based therapies. Current approaches require complex assistant molecules for combined release. To overcome the restrictions on the materials and environment, a novel and versatile DNA release method using a nano-electromechanical (NEMS) hypersonic resonator of gigahertz (GHz) frequency is developed. Results The micro-vortexes excited by ultra-high frequency acoustic wave can generate tunable shear stress at solid–liquid interface, thereby disrupting molecular interactions in immobilized multilayered polyelectrolyte thin films and releasing embedded DNA strands in a controlled fashion. Both finite element model analysis and experiment results verify the feasibility of this method. The release rate and released amount are confirmed to be well tuned. Owing to the different forces generated at different depth of the films, release of two types of DNA molecules with different velocities is achieved, which further explores its application in combined gene therapy. Conclusions Our research confirmed that this novel platform based on a nano-electromechanical hypersonic resonator works well for controllable single and multi-DNA release. In addition, the unique features of this resonator such as miniaturization and batch manufacturing open its possibility to be developed into a high-throughput, implantable and site targeting DNA release and delivery system.
... Recently, Yeo and coworkers demonstrated device architectures that utilize surface acoustic waves at high frequencies (>10 MHz) to suppress cavitation for gene delivery to human embryonic kidney cells and porcine tissue (27,28). Zhang et al. explored even higher frequencies (gigahertz) with bulk acoustic resonators and demonstrated intracellular delivery of doxorubicin and plasmids with high efficiency (29). As such, there is great promise in applying acoustic-based systems toward intracellular delivery to therapeutic and disease-relevant cell types (e.g., T cells, stem cells) with high throughput. ...
Article
Significance Commercial strategies to deliver biomolecular cargo ex vivo (e.g., electroporation, lipofection) to clinically relevant cell lines are limited by toxicity, cost, and throughput. These technical limitations have inhibited development of these technologies into streamlined clinical platforms for manufacturing gene-modified stem cells and cancer immunotherapies. Here, we demonstrate an acoustofluidic platform capable of delivering plasmids with high throughput to human T lymphocytes, peripheral blood mononuclear cells, and CD34 ⁺ hematopoietic stem and progenitor cells. Acoustofluidic-treated cells showed evidence of cytosolic DNA delivery, endocytic DNA aggregation, and nuclear membrane rupture. Collectively, these observations demonstrate the utility of this method as a research tool for gene editing applications and mechanistic studies of plasma membrane and nuclear membrane repair.
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The internalization of therapeutic molecules into cells-a critical step in enabling a suite of autologous ex vivo gene and cell therapies-is highly regulated by the lipid barrier imposed by the cell membrane. Strategies to increase the efficiency of delivering these exogenous payloads into the cell, while maintaining the integrity of both the therapeutic molecules to be delivered as well as the host cells they are delivered to, are therefore required. This is especially the case for suspension cells that are particularly difficult to transfect. In this work, we show that it is possible to enhance the uptake of short interfering RNA (siRNA) into nonadherent Jurkat and HuT 78 cells with a rapid poration-free method involving high-frequency (MHz order) acoustic excitation. The 2-fold enhancement in gene knockdown is almost comparable with that obtained with conventional nucleofection, which is among the most widely used intracellular delivery methods, but with considerably higher cell viabilities (>91% compared to approximately 76%) owing to the absence of pore formation. The rapid and effective delivery afforded by the platform, together with its low cost and scalability, therefore renders it a potent tool in the cell engineering pipeline.
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Chapter
Genome sequencing led to thousands of genes to study and their molecular cloning to provide ORF collection plasmids. The main approach to study their function involves analysis of the biological consequences of their expression or knockdown, in a cellular context. Given that, the starting point of such experiments is the delivery of the exogenous material, including plasmid DNA in cells. During the last decades, efforts were made to develop efficient methods and protocols to achieve this goal. The present chapter will first give a rapid overview of the main DNA transfer methods described so far: physical, chemical, and biological. Secondly, it will focus on the different methods having reached high-throughput nowadays. Finally, it will discuss the perspectives of this field in terms of future enhancements.
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We present a nanoscale acoustofluidic trap (AFT) which manipulates nanoparticles in a microfluidic system actuated by a gigahertz acoustic resonator. The AFT generates independent standing closed vortices with high-speed rotation. By carefully designing and optimizing the geometric confinements, the AFT is able to effectively capture and enrich sub-100 nm nanoparticles with low power consumption (0.25~5 μW/μm2) and rapid trapping (within 30 s), showing greatly enhanced particle operating ability towards its acoustic and optical counterparts. Using specifically functionalized nanoparticles (SFNPs) to selectively capture target molecules from the sample, the AFT produces a molecular concentration enhancement of ~200 times. We investigated the feasibility of the SFNPs-assisted AFT preconcentration method for biosensing applications, and successfully demonstrated its capability for serum prostate specific antigen (PSA) detection. The AFT is prepared with a fully CMOS-compatible process, and thus can be conveniently integrated on a single chip, with potential for “lab-on-a-chip” or point-of-care (POC) nanoparticle-based biosensing applications.
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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.
Chapter
The use of nanomaterials for drug delivery and cancer therapy has well established itself over the past decades. Compared with free chemotherapeutics, nanoparticle drug delivery systems achieve better performance in various areas, such as enhanced bioavailability, extended blood circulation time, and systemic control of drug release. Furthermore, coupling stimuli-activated functionality with these nanocomposites provides them with superior advantages in bypassing biological obstacles, maximizing the therapeutic efficacy, and minimizing the side effects of loaded drugs. Because of the active metabolism of tumor cells, the tumor microenvironment exhibits several distinguishing properties (e.g., acidic pH, over-expressed enzyme, hypoxia, and reductive environment) compared with normal tissues or cells, which has been a key rationale for the development of stimuli-activated nanomaterials for cancer therapy. In this chapter, we summarized the recent advances in various tumor microenvironment and intracellular signal-activated nanocomposites for anticancer drug delivery. Future perspectives on design consideration were also discussed in detail.
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MEMS/NEMS resonators are widely studied in biological detection, physical sensing, and quantum coupling. This paper reviews the latest research progress of MEMS/NEMS resonators with different structures. The resonance performance, new test method, and manufacturing process of single or double-clamped resonators, and their applications in mass sensing, micromechanical thermal analysis, quantum detection, and oscillators are introduced in detail. The material properties, resonance mode, and application in different fields such as gyroscope of the hemispherical structure, microdisk structure, drum resonator are reviewed. Furthermore, the working principles and sensing methods of the surface acoustic wave and bulk acoustic wave resonators and their new applications such as humidity sensing and fast spin control are discussed. The structure and resonance performance of tuning forks are summarized. This article aims to classify resonators according to different structures and summarize the working principles, resonance performance, and applications.
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Intracellular delivery is essential to therapeutic applications such as genome engineering and disease diagnosis. Current methods lack simple, non-invasive strategies, and are often hindered by long incubation time or high toxicity. Hydrodynamic approaches offer rapid and controllable delivery of small molecules, but thus far have not been demonstrated for delivering functional proteins. In this work, we developed a robust hydrodynamic approach based on gigahertz (GHz) acoustics to achieve rapid and non-invasive cytosolic delivery of biologically active proteins. With this method, GHz-based acoustic devices trigger oscillations through a liquid medium (acoustic streaming) generating shear stress on the cell membrane and inducing transient nanoporation. This mechanical effect enhances membrane permeability and enables cytosolic access to cationic proteins without disturbing their bioactivity. We evaluated the versatility of this approach through delivery of cationic fluorescent proteins to a range of cell lines, all of which displayed equally efficient delivery speed (≤ 20 minutes). Delivery of multiple enzymatically active proteins with functionality related to apoptosis or genetic recombination further demonstrated the relevance of this method.
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Surface-mediated gene delivery attracts more and more attentions in biomedical researches and applications due to its characteristics of low toxicity and localized delivery. Herein, a novel visible-light regulated surface-mediated gene delivery platform is exhibited, arising from the photo-induced surface charge accumulation on silicon. Silicon with a pn junction is used, and tested subsequently for the behavior of surface-mediated gene delivery under visible-light illumination. It is found that positive charge accumulation under light illumination changes the surface potential, and then facilitates the delivery of gene-loaded carriers. As a result, the gene expression efficiency shows a significant improvement from 6% to 28% under a 10 min visible-light illumination. Such improvement is ascribed to that the increased surface potential caused by light illumination, which promotes both the release of gene-loaded carriers and the cellular uptake. This work suggests that silicon with photovoltaic effect could offer a new strategy for surface-mediated gene delivery related biomedical researches and applications.
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The development of rapid and efficient tools to modulate neurons is vital for the treatment of nervous system diseases. Here, a novel non-invasive neurite outgrowth modulation method based on a controllable acoustic streaming effect induced by an electromechanical gigahertz resonator microchip is reported. The results demonstrate that the gigahertz acoustic streaming can induce cell structure changes within a 10 min period of stimulation, which promotes a high proportion of neurite bearing cells and encourages longer neurite outgrowth. Specifically, the resonator stimulation not only promotes outgrowth of neurites, but also can be combined with chemical mediated methods to accelerate the direct entry of nerve growth factor (NGF) into cells, resulting in higher modulation efficacy. Owing to shear stress caused by the acoustic streaming effect, the resonator microchip mediates stress fiber formation and induces the neuron-like phenotype of PC12 cells. We suggest that this method may potentially be applied to precise single-cell modulation, as well as in the development of non-invasive and rapid disease treatment strategies.
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Tracing magnetically labeled cells with magnetic resonance imaging (MRI) is an emerging and promising approach to uncover in vivo behaviors of cells in cell therapy. Today, existing methods for the magnetic labeling of cells are cumbersome and time-consuming, which has greatly limited the progress of such studies on cell therapy. Thus, in this study, using the flow cytometric loading technology, we develop a sonoporation-based microfluidic chip (i.e. a microfluidic chip integrated with ultrasound; MCU), to achieve the safe, instant, convenient and continuous magnetic labeling of cells. For the MCU we designed, a suitable group of operating conditions for safely and efficiently loading superparamagnetic iron oxide (SPIO) nanoparticles into DC2.4 cells was identified experimentally. Under the identified operating conditions, the DC2.4 cells could be labeled in approximately 2 min with high viability (94%) and a high labeling quantity of SPIO nanoparticles (19 pg of iron per cell). In addition, the proliferative functions of the cells were also well maintained after labeling. Furthermore, the in vivo imaging ability of the DC2.4 cells labeled using the MCU was verified by injecting the labeled cells into the leg muscle of the C57BL/6 mice. The results show that the excellent imaging outcome can be continuously achieved for 7 days at a density of 106 cells/mL. This work can provide insight for the design of magnetic cell labeling devices and promote the MRI-based study of cell therapies.
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The hydrodynamic method mimics the in vivo environment of the mechanical effect on cell stimulation, which not only modulates cell physiology but also shows excellent intracellular delivery ability. Herein, a hydrodynamic intracellular delivery system based on the gigahertz acoustic streaming (AS) effect is proposed, which presents powerful targeted delivery capabilities with high efficiency and universality. Results indicate that the range of cells with AuNR introduction is related to that of AS, enabling a tunable delivery range due to the adjustability of the AS radius. Moreover, with the assistance of AS, the organelle localization delivery of AuNRs with different modifications is enhanced. AuNRs@RGD is inclined to accumulate in the nucleus, while AuNRs@BSA tend to enter the mitochondria and AuNRs@PEGnK tend to accumulate in the lysosome. Finally, the photothermal effect is proved based on the large quantities of AuNRs introduced via AS. The abundant introduction of AuNRs under the action of AS can achieve rapid cell heating with the irradiation of a 785 nm laser, which has great potential in shortening the treatment cycle of photothermal therapy (PTT). Thereby, an efficient hydrodynamic technology in AuNR introduction based on AS has been demonstrated. The outstanding location delivery and organelle targeting of this method provides a new idea for precise medical treatment.
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The mechanical and electrical properties of biomaterials are essential in cell function regulation during cell-biomaterial interaction. However, previous studies focused on probing cell regulation mechanisms under one type of stimulus, and a platform that enables the study of electromechanical coupling effects of a biomaterial on cells is still lacking. Here, we present an in-situ electromechanical testing and loading system to image live cells when co-cultured with electroactive biomaterials. The system can provide accurate and repeatable stretch on biomaterials and cells to mimic in vivo tension microenvironment. Besides, the integrated displacement transducer, force sensor, and electrical signal detector enable the real time detection of electromechanical signals on electroactive biomaterials under various stretch loading. Combined with a microscope, live cell imaging can be realized to probe cell behavior. The feasibility of the system is validated by culturing mesenchymal stem cells on piezoelectric nanofiber and conductive hydrogel. Experiment results show the device as a reliable and accurate tool to investigate electromechanical properties of biomaterials and probe essential features of live cells. Our system provides a way to correlate cell behavior with electromechanical cues directly and is useful for exploration of cell function during cell-biomaterial interaction.
Conference Paper
This work reports a novel acoustic resonator system integrated dual functions of biological samples capture and amount monitoring on a single chip. The system could capture samples from nano-sized proteins to micro-sized cells on micro-sized chip precisely with controllable concentration, meanwhile the high sensitivity mass sensing was achieved during the capture process. The devices were further applied to study the cell growth and cytotoxicity. Results indicated that it was possible to capture and monitor the physiological changes in a single cell level. This work explores a new opportunity on the development of miniaturized multiplex biosensing devices on a single chip.
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Here, we present a high performance uncooled near-infrared (NIR) detector comprising of a giga hertz (GHz) solidly mounted resonator (SMR) and gold nanorods (GNRs) arrays. By coupling the localized surface plasmon resonances of GNRs, the resonator system exhibits optimized optical response to vis-NIR region. Both simulation and experiments demonstrate the hybrid GNRs-SMR exhibit significantly enhanced optical responsive sensitivity of NIR, the tunable aspect ratios (AR) of GNRs enable resonator respond sensitively to selected light. Specially, taking advantage of the acoustofluidic effect of SMR, the GNRs can be controllably and precisely modified on the microchip surface in an ultra-short time, which addresses one of the most fundamental challenges in the localized functionalization of micro/nano scale surface. The presented work opens new directions in development of novel miniaturized, tunable NIR detector.
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Ultrasound constitutes a powerful means for materials processing. Similarly, a new field has emerged demonstrating the possibility for harnessing sound energy sources at considerably higher frequencies (10 MHz to 1 GHz) compared to conventional ultrasound (⩽3 MHz) for synthesizing and manipulating a variety of bulk, nanoscale, and biological materials. At these frequencies and the typical acoustic intensities employed, cavitation—which underpins most sonochemical or, more broadly, ultrasound‐mediated processes—is largely absent, suggesting that altogether fundamentally different mechanisms are at play. Examples include the crystallization of novel morphologies or highly oriented structures; exfoliation of 2D quantum dots and nanosheets; polymer nanoparticle synthesis and encapsulation; and the possibility for manipulating the bandgap of 2D semiconducting materials or the lipid structure that makes up the cell membrane, the latter resulting in the ability to enhance intracellular molecular uptake. These fascinating examples reveal how the highly nonlinear electromechanical coupling associated with such high‐frequency surface vibration gives rise to a variety of static and dynamic charge generation and transfer effects, in addition to molecular ordering, polarization, and assembly—remarkably, given the vast dimensional separation between the acoustic wavelength and characteristic molecular length scales, or between the MHz‐order excitation frequencies and typical THz‐order molecular vibration frequencies.
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Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9 (CRISPR/Cas9) has transformed our ability to edit the human genome selectively. This technology has quickly become the most standardized and reproducible gene editing tool available. Catalyzing rapid advances in biomedical research and genetic engineering, the CRISPR/Cas9 system offers great potential to provide diagnostic and therapeutic options for the prevention and treatment of currently incurable single-gene and more complex human diseases. However, significant barriers to the clinical application of CRISPR/Cas9 remain. While in vitro, ex vivo, and in vivo gene editing has been demonstrated extensively in a laboratory setting, the translation to clinical studies is currently limited by shortfalls in the precision, scalability, and efficiency of delivering CRISPR/Cas9-associated reagents to their intended therapeutic targets. To overcome these challenges, recent advancements manipulate both the delivery cargo and vehicles used to transport CRISPR/Cas9 reagents. With the choice of cargo informing the delivery vehicle, both must be optimized for precision and efficiency. This review aims to summarize current bioengineering approaches to applying CRISPR/Cas9 gene editing tools towards the development of emerging cellular therapeutics, focusing on its two main engineerable components: the delivery vehicle and the gene editing cargo it carries. The contemporary barriers to biomedical applications are discussed within the context of key considerations to be made in the optimization of CRISPR/Cas9 for widespread clinical translation.
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Almost 2 decades ago, it was pointed out that physical therapists tended to overlook the tenuous nature of the scientific basis for the use of therapeutic ultrasound. The purpose of this review is to examine the literature regarding the biophysical effects of therapeutic ultrasound to determine whether these effects may be considered sufficient to provide a reason (biological rationale) for the use of insonation for the treatment of people with pain and soft tissue injury. This review does not discuss articles that examined the clinical usefulness of ultrasound (see article by Robertson and Baker titled “A Review of Therapeutic Ultrasound: Effectiveness Studies” in this issue). The frequently described biophysical effects of ultrasound either do not occur in vivo under therapeutic conditions or have not been proven to have a clinical effect under these conditions. This review reveals that there is currently insufficient biophysical evidence to provide a scientific foundation for the clinical use of therapeutic ultrasound for the treatment of people with pain and soft tissue injury.
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Intracellular delivery of materials has become a critical component of genome-editing approaches, ex vivo cell-based therapies, and a diversity of fundamental research applications. Limitations of current technologies motivate development of next-generation systems that can deliver a broad variety of cargo to diverse cell types. Here we review in vitro and ex vivo intracellular delivery approaches with a focus on mechanisms, challenges and opportunities. In particular, we emphasize membrane-disruption-based delivery methods and the transformative role of nanotechnology, microfluidics and laboratory-on-chip technology in advancing the field. © 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
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Culture of cells using various microfluidic devices is becoming more common within experimental cell biology. At the same time, a technological radiation of microfluidic cell culture device designs is currently in progress. Ultimately, the utility of microfluidic cell culture will be determined by its capacity to permit new insights into cellular function. Especially insights that would otherwise be difficult or impossible to obtain with macroscopic cell culture in traditional polystyrene dishes, flasks or well-plates. Many decades of heuristic optimisation have gone into perfecting conventional cell culture devices and protocols. In comparison, even for the most commonly used microfluidic cell culture devices, such as those fabricated from polydimethylsiloxane (PDMS), collective understanding of the differences in cellular behaviour between microfluidic and macroscopic culture is still developing. Moving in vitro culture from macroscopic culture to PDMS based devices can come with unforeseen challenges. Changes in device material, surface coating, cell number per unit surface area or per unit media volume may all affect the outcome of otherwise standard protocols. In this review, we outline some of the advantages and challenges that may accompany a transition from macroscopic to microfluidic cell culture. We focus on decisive factors that distinguish macroscopic from microfluidic cell culture to encourage a reconsideration of how macroscopic cell culture principles might apply to microfluidic cell culture.
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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.
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Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations, including their reliance on exogenous materials or electrical fields, which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30-80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to 11 cell types, including embryonic stem cells and immune cells. When used for the delivery of transcription factors, the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed, its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications.
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We demonstrate the integration of vertically aligned carbon nanofibre (VACNF) elements with the intracellular domains of viable cells for controlled biochemical manipulation. Deterministically synthesized VACNFs were modified with either adsorbed or covalently-linked plasmid DNA and were subsequently inserted into cells. Post insertion viability of the cells was demonstrated by continued proliferation of the interfaced cells and long-term (> 22 day) expression of the introduced plasmid. Adsorbed plasmids were typically desorbed in the intracellular domain and segregated to progeny cells. Covalently bound plasmids remained tethered to nanofibres and were expressed in interfaced cells but were not partitioned into progeny, and gene expression ceased when the nanofibre was no longer retained. This provides a method for achieving a genetic modification that is non-inheritable and whose extent in time can be directly and precisely controlled. These results demonstrate the potential of VACNF arrays as an intracellular interface for monitoring and controlling subcellular and molecular phenomena within viable cells for applications including biosensors, in vivo diagnostics, and in vivo logic devices.
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Most present nanodrug delivery systems have been developed to target cancer cells but rarely nuclei. However, nuclear-targeted drug delivery is expected to kill cancer cells more directly and efficiently. In this work, TAT peptide has been employed to conjugate onto mesoporous silica nanoparticles (MSNs-TAT) with high payload for nuclear-targeted drug delivery for the first time. Monodispersed MSNs-TAT of varied particle sizes have been synthesized to investigate the effects of particle size and TAT conjugation on the nuclear membrane penetrability of MSNs. MSNs-TAT with a diameter of 50 nm or smaller can efficiently target the nucleus and deliver the active anticancer drug doxorubicin (DOX) into the targeted nucleus, killing these cancer cells with much enhanced efficiencies. This study may provide an effective strategy for the design and development of cell-nuclear-targeted drug delivery.
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Sonoporation is a useful biophysical mechanism for facilitating the transmembrane delivery of therapeutic agents from the extracellular to the intracellular milieu. Conventionally, sonoporation is carried out in the presence of ultrasound contrast agents, which are known to greatly enhance transient poration of biological cell membranes. However, in vivo contrast agents have been observed to induce capillary rupture and haemorrhage due to endothelial cell damage and to greatly increase the potential for cell lysis in vitro. Here, we demonstrate sonoporation of cardiac myoblasts in the absence of contrast agent (CA-free sonoporation) using a low-cost ultrasound-microfluidic device. Within this device an ultrasonic standing wave was generated, allowing control over the position of the cells and the strength of the acoustic radiation forces. Real-time single-cell analysis and retrospective post-sonication analysis of insonated cardiac myoblasts showed that CA-free sonoporation induced transmembrane transfer of fluorescent probes (CMFDA and FITC-dextran) and that different mechanisms potentially contribute to membrane poration in the presence of an ultrasonic wave. Additionally, to the best of our knowledge, we have shown for the first time that sonoporation induces increased cell cytotoxicity as a consequence of CA-free ultrasound-facilitated uptake of pharmaceutical agents (doxorubicin, luteolin, and apigenin). The US-microfluidic device designed here provides an in vitro alternative to expensive and controversial in vivo models used for early stage drug discovery, and drug delivery programs and toxicity measurements.
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Many transfection techniques can deliver biomolecules into cells, but the dose cannot be controlled precisely. Delivering well-defined amounts of materials into cells is important for various biological studies and therapeutic applications. Here, we show that nanochannel electroporation can deliver precise amounts of a variety of transfection agents into living cells. The device consists of two microchannels connected by a nanochannel. The cell to be transfected is positioned in one microchannel using optical tweezers, and the transfection agent is located in the second microchannel. Delivering a voltage pulse between the microchannels produces an intense electric field over a very small area on the cell membrane, allowing a precise amount of transfection agent to be electrophoretically driven through the nanochannel, the cell membrane and into the cell cytoplasm, without affecting cell viability. Dose control is achieved by adjusting the duration and number of pulses. The nanochannel electroporation device is expected to have high-throughput delivery applications.
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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.
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The interaction of laser-generated tandem microbubble (maximum diameter of about 50  μm) with single (rat mammary carcinoma) cells is investigated in a 25-μm liquid layer. Antiphase and coupled oscillation of the tandem microbubble leads to the formation of alternating, directional microjets (with max microstreaming velocity of 10  m/s) and vortices (max vorticity of 350 000  s{-1}) in opposite directions. Localized and directional membrane poration (200 nm to 2  μm in pore size) can be produced by the tandem microbubble in an orientation and proximity-dependent manner, which is absent from a single oscillating microbubble of comparable size and at the same stand-off distance.
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A generalized platform for introducing a diverse range of biomolecules into living cells in high-throughput could transform how complex cellular processes are probed and analyzed. Here, we demonstrate spatially localized, efficient, and universal delivery of biomolecules into immortalized and primary mammalian cells using surface-modified vertical silicon nanowires. The method relies on the ability of the silicon nanowires to penetrate a cell's membrane and subsequently release surface-bound molecules directly into the cell's cytosol, thus allowing highly efficient delivery of biomolecules without chemical modification or viral packaging. This modality enables one to assess the phenotypic consequences of introducing a broad range of biological effectors (DNAs, RNAs, peptides, proteins, and small molecules) into almost any cell type. We show that this platform can be used to guide neuronal progenitor growth with small molecules, knock down transcript levels by delivering siRNAs, inhibit apoptosis using peptides, and introduce targeted proteins to specific organelles. We further demonstrate codelivery of siRNAs and proteins on a single substrate in a microarray format, highlighting this technology's potential as a robust, monolithic platform for high-throughput, miniaturized bioassays.
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Electric impulses (8 kV/cm, 5 microseconds) were found to increase greatly the uptake of DNA into cells. When linear or circular plasmid DNA containing the herpes simplex thymidine kinase (TK) gene is added to a suspension of mouse L cells deficient in the TK gene and the cells are then exposed to electric fields, stable transformants are formed that survive in the HAT selection medium. At 20 degrees C after the application of three successive electric impulses followed by 10 min to allow DNA entry there result 95 (+/- 3) transformants per 10(6) cells and per 1.2 micrograms DNA. Compared with biochemical techniques, the electric field method of gene transfer is very simple, easily applicable, and very efficient. Because the mechanism of DNA transport through cell membranes is not known, a simple physical model for the enhanced DNA penetration into cells in high electric fields is proposed. According to this ' electroporation model' the interaction of the external electric field with the lipid dipoles of a pore configuration induces and stabilizes the permeation sites and thus enhances cross membrane transport.
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We report gene transfer to the Edinburgh insertional mutant mouse (cf/cf), delivering CFTR cDNA-liposome complexes into the airways by nebulization. We show full restoration of cAMP related chloride responses in some animals and demonstrate, in the same tissues, human CFTR cDNA expression. Overall, a range of correction was seen with restoration of about 50% of the deficit between wild type mice and untreated cf/cf controls. We report modest correction in the intestinal tract following direct instillation and provide initial encouraging safety data for both the respiratory and intestinal tract following the liposome mediated gene delivery. The non-viral nature and potentially lower immunogenicity of DNA-liposomes suggest that this may offer a therapeutic alternative to adenoviral therapies.
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Efficient DNA transfection is critical for biological research and new clinical therapies, but the mechanisms responsible for DNA uptake are unknown. Current nonviral transfection methods, empirically designed to maximize DNA complexation and/or membrane fusion, are amenable to enhancement by a variety of chemicals. These chemicals include particulates, lipids, and polymer complexes that optimize DNA complexation/condensation, membrane fusion, endosomal release, or nuclear targeting, which are the presumed barriers to gene delivery. Most chemical enhancements produce a moderate increase in gene delivery and a limited increase in gene expression. As a result, the efficiency of transfection and level of gene expression after nonviral DNA delivery remain low, suggesting the existence of additional unidentified barriers. Here, we tested the hypothesis that DNA transfection efficiency is limited by a simple physical barrier: low DNA concentration at the cell surface. We used dense silica nanoparticles to concentrate DNA-vector (i.e. DNA-transfection reagent) complexes at the surface of cell monolayers; manipulations that increased complex concentration at the cell surface enhanced transfection efficiency by up to 8.5-fold over the best commercially available transfection reagents. We predict that manipulations aimed at optimizing DNA complexation or membrane fusion have a fundamental physical limit; new methods designed to increase transfection efficiency must increase DNA concentration at the target cell surface without adding to the toxicity.
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Considered by some to be among the simpler forms of life, viruses represent highly evolved natural vectors for the transfer of foreign genetic information into cells. This attribute has led to extensive attempts to engineer recombinant viral vectors for the delivery of therapeutic genes into diseased tissues. While substantial progress has been made, and some clinical successes are over the horizon, further vector refinement and/or development is required before gene therapy will become standard care for any individual disorder.
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Almost 2 decades ago, it was pointed out that physical therapists tended to overlook the tenuous nature of the scientific basis for the use of therapeutic ultrasound. The purpose of this review is to examine the literature regarding the biophysical effects of therapeutic ultrasound to determine whether these effects may be considered sufficient to provide a reason (biological rationale) for the use of insonation for the treatment of people with pain and soft tissue injury. This review does not discuss articles that examined the clinical usefulness of ultrasound (see article by Robertson and Baker titled "A Review of Therapeutic Ultrasound: Effectiveness Studies" in this issue). The frequently described biophysical effects of ultrasound either do not occur in vivo under therapeutic conditions or have not been proven to have a clinical effect under these conditions. This review reveals that there is currently insufficient biophysical evidence to provide a scientific foundation for the clinical use of therapeutic ultrasound for the treatment of people with pain and soft tissue injury.
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The challenge for successful delivery of foreign DNA into cells in vitro, a key technique in cell and molecular biology with important biomedical implications, is to improve transfection efficiency while leaving the cell's architecture intact. Here we show that a variety of mammalian cells can be directly transfected with DNA without perturbing their structure by first creating a tiny, localized perforation in the membrane using ultrashort (femtosecond), high-intensity, near-infrared laser pulses. Not only does this superior optical technique give high transfection efficiency and cell survival, but it also allows simultaneous evaluation of the integration and expression of the introduced gene.
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The major advantages of "naked DNA gene therapy" are its simplicity and a low or negligible immune response. Gene delivery by DNA electroporation (EP) involves injection of DNA and the application of a brief electric pulse to enhance cellular permeability. Although EP is an efficient gene transduction technique in rodents, it requires much higher voltages (>500 V) in larger animals, and hence, in practice it would be hazardous for human patients, as it would cause serious tissue damage. To overcome the obstacles associated with EP-mediated gene delivery in vivo, we developed a new method of gene transduction that uses laser energy. The femtosecond infrared titanium sapphire laser beam was developed specifically for enhancing in vivo gene delivery without risks of tissue damage. System optimization revealed that injection of 10 micro g naked DNA into the tibial muscle of mice followed by application of the laser beam for 5 s, focused to 2 mm depth upon an area of 95 x 95 micro m(2), resulted in the highest intensity and duration of gene expression with no histological or biochemical evidence of muscle damage. We assessed the potential clinical application of LBGT technology by using it to transfer the murine erythropoietin (mEpo) gene into mice. LBGT-mediated mEpo gene delivery resulted in elevated (>22%) hematocrit levels that were sustained for 8 weeks. Gene expression following LBGT was detected for >100 days. Hence, LBGT is a simple, safe, effective, and reproducible method for therapeutic gene delivery with significant clinical potential.
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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.
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Nonviral gene delivery is a promising, safe, therapeutic tool in regenerative medicine. This study is the first to achieve nonviral, ultrasound-based, osteogenic gene delivery that leads to bone tissue formation, in vivo. We hypothesized that direct in vivo sonoporation of naked DNA encoding for the osteogenic gene, recombinant human bone morphogenetic protein-9 (rhBMP-9) would induce bone formation. A luciferase plasmid (Luc), encoding rhBMP-9 or empty pcDNA3 vector mixed with microbubbles, was injected into the thigh muscles of mice. After injection, noninvasive sonoporation was applied. Luc activity was monitored noninvasively, and quantitatively using bioluminescence imaging in vivo, and found for 14 days with a peak expression on day 7. To examine osteogenesis in vivo, rhBMP-9 plasmid was sonoporated into the thigh muscles of transgenic mice that express the Luc gene under the control of a human osteocalcin promoter. Following rhBMP-9 sonoporation, osteocalcin-dependent Luc expression lasted for 24 days and peaked on day 10. Bone tissue was formed in the site of rhBMP-9 delivery, as was shown by micro-computerized tomography and histology. The sonoporation method was also compared with previously developed electrotransfer-based gene delivery and was found significantly inferior in its efficiency of gene delivery. We conclude that ultrasound-mediated osteogenic gene delivery could serve as a therapeutic solution in conditions requiring bone tissue regeneration after further development that will increase the transfection efficiency.
Article
DOI:https://doi.org/10.1103/PhysRevLett.2.298
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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.
Article
Non-destructive introduction of genes, proteins, and small molecules into mammalian cells with high efficiency is a challenging, yet critical process. Here we demonstrate a simple nano-electroporation platform to achieve highly efficient molecular delivery and high transfection yields with excellent uniformity and cell viability. The system is built on alumina nanostraws extending from a track-etched membrane, forming an array of hollow nanowires connected to an underlying microfluidic channel. Cellular engulfment of the nanostraws provides an intimate contact, significantly reducing the necessary electroporation voltage and increasing homogeneity over a large area. Biomolecule delivery is achieved by diffusion through the nanostraws and enhanced by electrophoresis during pulsing. The system was demonstrated to offer excellent spatial, temporal, and dose control for delivery, as well as providing high-yield co-transfection and sequential transfection.
Article
Introduction: With a wealth of knowledge on the effect of nanoparticle properties, including size, shape, charge and composition, on intracellular delivery, little has been reported on the effect of the cell cycle on the intracellular delivery and activity of nanomedicines including non-viral gene delivery systems. The aim of this review is to shed a light on this topic. Areas covered: It is now evident that nanoparticle cell uptake varies with the cell cycle phase. This review addresses this variation by dissecting the effect of cell population heterogeneity on the intracellular delivery and activity of nanomedicines with a special focus on non-viral gene delivery and combination therapy modalities that utilize cell cycle inhibitors as co-targets for therapy. In addition, the importance of three-dimensional (3D) culture systems in the drug delivery field within the context of the cell cycle will be addressed. Expert opinion: The understanding of the cell cycle machinery has improved dramatically over the last few decades. Developing combination therapy modalities that target the cell cycle to achieve better cancer patient outcome should now be the focus. Furthermore, more effort should be placed on developing a reliable, consistent, high throughput 3D cell culture system since these systems more closely resemble the cell cycle status of in vivo tumors. A switch from 2D to 3D culture systems, to more accurately predict the in vivo efficacy of nanoparticle drug delivery systems, is desirable.
Article
This paper demonstrates membrane poration of a single suspension cell due to a fast liquid microjet. The jet is formed during the collapse of a laser induced bubble created at a variable stand-off distance from the target cell. The cell is trapped by a converging structure within a microfluidic chip. The asymmetrical growth and collapse of the cavitation bubble next to the cell lead to the microjetting, which deforms and porates the cell membrane. In the experiments, the membrane porations of myeloma cells are probed with the uptake of trypan blue. Time-resolved studies of the diffusion of trypan blue show a marked dependency on the bubble dynamics, i.e. the stand-off distance. The penetration length of the dye increases with shorter distances. Numerical simulations of the diffusion process agree with larger pores formed on the cell membrane. This method allows for a fast, repeatable, and localized rupture of membranes of individual cells in suspension.
Article
A retroviral vector system based on the human immunodeficiency virus (HIV) was developed that, in contrast to a murine leukemia virus-based counterpart, transduced heterologous sequences into HeLa cells and rat fibroblasts blocked in the cell cycle, as well as into human primary macrophages. Additionally, the HIV vector could mediate stable in vivo gene transfer into terminally differentiated neurons. The ability of HIV-based viral vectors to deliver genes in vivo into nondividing cells could increase the applicability of retroviral vectors in human gene therapy.
Article
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.
Article
Acoustic streaming is a stationary fluid motion induced by intense ultrasound. This paper describes the mechanism of generation of acoustic streaming, theoretically and experimentally. A qualitative explanation in relation to acoustic radiation pressure is followed by a detailed discussion based on the formulation of the phenomenon. The spatial non-uniformity of the sound field and energy dissipation due to viscosity are requisite for the generation of streaming. In the development of streaming, two kinds of nonlinearity are important: one is fluid-dynamic nonlinearity, which accounts for effects of inertial force compared to viscous force in fluid motion, and the other is acoustic nonlinearity, which determines the driving force of streaming. © 1998 Scripta Technica, Electron Comm Jpn Pt 3, 81(10): 1–8, 1998
Article
Electroporation or electropermeabilization is one of the most powerful biological techniques in cell studies. Applying the high voltage electric field in vicinity of the cells can generate nanopores in cell membrane. Varying with the intensity and the duration of these applied electric field, the created nanopores can be temporary (reversible electroporation) or permanent (irreversible electroporation). Reversible electroporation is usually conducted to insert biological samples into the cells. Cells are also electroporated irreversibly to release their intercellular contents for further biological investigations. In comparison with the conventional electroporation devices, microfluidic (microscale) electroporation devices have some advantages such as higher cell viability rate, high transfection efficiency, lower sample contamination, and smaller Joule heating effect. In this article, the latest advancement in microfluidic cell electroporation is reviewed. First, the underlying theory of membrane permeabilization is reviewed and the leading analytical studies on the cell electroporation are presented. Following that, different experimental methods are compared. Finally, some suggestions are proposed for the future studies. KeywordsElectroporation–Cell–Microfluidics–Lab-on-a-chip
Electroporation is a membrane phenomenon which involves fundamental behavior of cell and artificial bilayer membranes, and increasingly attracts consideration for applications in biology, biotechnology and medicine. Understanding of the basic mechanisms underlying electroporation is therefore important, and provides the motivation of this review of the essential features of theoretical models of electroporation. We particularly emphasize the ability of these models to describe experimental results. Here we discuss the theoretical models that have been proposed, their underlying assumptions, and their successes and failures. Most of our emphasis is on transient aqueous pore models, which can account for: (1) key features of mechanical instability (irreversible consequence of electroporation) in planar lipid bilayers at elevated voltages, (2) dramatic reversible electrical behavior of certain planar membranes and of cell membranes, and (3) some features of molecular transport. In contrast, theories which do not explicitly treat pores appear unable to account for key electroporation phenomena, and are only briefly discussed.
Article
Polymers have played an integral role in the advancement of drug delivery technology by providing controlled release of therapeutic agents in constant doses over long periods, cyclic dosage, and tunable release of both hydrophilic and hydrophobic drugs. From early beginnings using off-the-shelf materials, the field has grown tremendously, driven in part by the innovations of chemical engineers. Modern advances in drug delivery are now predicated upon the rational design of polymers tailored for specific cargo and engineered to exert distinct biological functions. In this review, we highlight the fundamental drug delivery systems and their mathematical foundations and discuss the physiological barriers to drug delivery. We review the origins and applications of stimuli-responsive polymer systems and polymer therapeutics such as polymer-protein and polymer-drug conjugates. The latest developments in polymers capable of molecular recognition or directing intracellular delivery are surveyed to illustrate areas of research advancing the frontiers of drug delivery.
Article
Nanomaterials are promising candidates to improve the delivery efficiency and control of active agents such as DNA or drugs directly into cells. Here we demonstrate cell-culture platforms of nanotemplated "nanostraws" that pierce the cell membrane, providing a permanent fluidic pipeline into the cell for direct cytosolic access. Conventional polymeric track-etch cell culture membranes are alumina coated and etched to produce fields of nanostraws with controllable diameter, thickness, and height. Small molecules and ions were successfully transported into the cytosol with 40 and 70% efficiency, respectively, while GFP plasmids were successfully delivered and expressed. These platforms open the way for active, reproducible delivery of a wide variety of species into cells without endocytosis.
Article
We report a microfluidic based approach for single cell microinjection in which fluid streams direct a cell onto a fixed microneedle in contrast to moving a microneedle towards an immobilized cell, as done in conventional methods. The approach simplifies microinjection and offers the potential for flow through automated microinjection of cells.
Article
Particulate delivery systems such as liposomes and polymeric nano- and microparticles are attracting great interest for developing new vaccines. Materials and formulation properties essential for this purpose have been extensively studied, but relatively little is known about the influence of the administration route of such delivery systems on the type and strength of immune response elicited. Thus, the present study aimed at elucidating the influence on the immune response when of immunising mice by different routes, such as the subcutaneous, intradermal, intramuscular, and intralymphatic routes with ovalbumin-loaded liposomes, N-trimethyl chitosan (TMC) nanoparticles, and poly(lactide-co-glycolide) (PLGA) microparticles, all with and without specifically selected immune-response modifiers. The results showed that the route of administration caused only minor differences in inducing an antibody response of the IgG1 subclass, and any such differences were abolished upon booster immunisation with the various adjuvanted and non-adjuvanted delivery systems. In contrast, the administration route strongly affected both the kinetics and magnitude of the IgG2a response. A single intralymphatic administration of all evaluated delivery systems induced a robust IgG2a response, whereas subcutaneous administration failed to elicit a substantial IgG2a response even after boosting, except with the adjuvanted nanoparticles. The intradermal and intramuscular routes generated intermediate IgG2a titers. The benefit of the intralymphatic administration route for eliciting a Th1-type response was confirmed in terms of IFN-gamma production of isolated and re-stimulated splenocytes from animals previously immunised with adjuvanted and non-adjuvanted liposomes as well as with adjuvanted microparticles. Altogether the results show that the IgG2a associated with Th1-type immune responses are sensitive to the route of administration, whereas IgG1 response associated with Th2-type immune responses were relatively insensitive to the administration route of the particulate delivery systems. The route of administration should therefore be considered when planning and interpreting pre-clinical research or development on vaccine delivery systems.
Article
Genetic modification of cells is a critical step involved in many cell therapy and gene therapy protocols. In these applications, cell samples of large volume (10(8)-10(9)cells) are often processed for transfection. This poses new challenges for current transfection methods and practices. Here we present a novel flow-through electroporation method for delivery of genes into cells at high flow rates (up to approximately 20 mL/min) based on disposable microfluidic chips, a syringe pump, and a low-cost direct current (DC) power supply that provides a constant voltage. By eliminating pulse generators used in conventional electroporation, we dramatically lowered the cost of the apparatus and improved the stability and consistency of the electroporation field for long-time operation. We tested the delivery of pEFGP-C1 plasmids encoding enhanced green fluorescent protein into Chinese hamster ovary (CHO-K1) cells in the devices of various dimensions and geometries. Cells were mixed with plasmids and then flowed through a fluidic channel continuously while a constant voltage was established across the device. Together with the applied voltage, the geometry and dimensions of the fluidic channel determined the electrical parameters of the electroporation. With the optimal design, approximately 75% of the viable CHO cells were transfected after the procedure. We also generalize the guidelines for scaling up these flow-through electroporation devices. We envision that this technique will serve as a generic and low-cost tool for a variety of clinical applications requiring large volume of transfected cells.
Article
Scrape loading and sonication loading are two recently described methods of introducing macromolecules into living cells. We have tested the efficacy of these methods for transfection of mammalian cells with exogenous DNA, using selection systems based either on resistance to the drug G418 (Geneticin) or on acquisition of the ability to utilize the salvage pathway of pyrimidine biosynthesis. These loading methods can be employed to generate cell lines that express the gene product of the transfected DNA molecules both transiently and stably. Optimal transfection is observed when the DNA is added to cells in physiological saline lacking divalent cations and containing K+ in place of Na+. DNA molecules 7.1 to 30 kilobases long have been introduced by the scrape loading procedure. In addition, the scrape loading procedure has been employed for cotransfection and subsequent expression of nonselectable genes encoded on DNA molecules added in a mixture with DNA molecules whose expression is selected. Cell lines expressing oncogenes or proteins that are important for regulation of cell growth and division have been obtained by this procedure. The scrape loading procedure is also useful for studies of the cellular changes that occur upon expression of an exogenous gene. As many as 80% of cells scrape loaded with the plasmid pC6, which encodes the simian virus 40 large tumor antigen, contained this protein in the nucleus between 1 and 5 days after transfection. Thus, scrape loading and sonication loading are simple, economical, and reproducible methods for introduction of DNA molecules into adherent and nonadherent cells, and these methods may be useful in the future for experimentation at both fundamental and applied levels.
Article
A minute hole upon a cultured cell, perforated with a finely focused laser beam, was found to repair itself within a short period of time. The procedure constitutes a new way of introducing exogenous gene materials dissolved in medium into cells. The 'laser-aided' DNA transfection is better than the existing methods because it allows the treatment of a large number of cells in a shorter time, and an improved success rate.
Article
Direct microinjection of DNA by glass micropipettes was used to introduce the Herpes simplex virus thymidine kinase gene into cultured mammalian cells. When DNA was delivered directly into the nuclei of LMTK-, a mouse cell line deficient in thymidine kinase activity, 50--100% of the cells expressed TK enzymatic activity. In contrast, no TK activity could be detected when the DNA was injected into the cytoplasm. The number of injected LMTK- cells capable of indefinite growth in a TK+ selective medium (that is, transformants) depended on the nature of the plasmid DNA into which the HSV-TK gene was inserted. One cell in 500-1000 cells which received nuclear injections with pBR322/TK DNA gave rise to a viable colony when grown in HAT medium (that is, a TK+ selective medium). The transformation frequency increased to one in five injected cells when specific SV40 DNA sequences were also introduced into the HSV-TK plasmid. With the microinjection procedure transformation frequency was relatively insensitive to DNA concentration and did not depend on co-injecting with a carrier DNA. Most of the transformants were stable in nonselective medium as soon as they could be tested.
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
Cultured Chinese hamster ovary cells were exposed to 2.25-MHz ultrasound in sterile 4.5-mL polyethylene chambers and tested for cell lysis, sonoporation and DNA transfection. Ten percent of Albunex, a gas-body-based ultrasound contrast agent, was added to ensure cavitation nucleation, and the chambers were rotated at 60 rpm to promote cavitation activity during the 1-min exposures. Uptake of large fluorescent dextran molecules by some cells was observed for spatial peak pressure amplitudes as low as 0.1 MPa, which indicates transient permeabilization and resealing, i.e., sonoporation, of these cells during exposure. Significant lysis occurred for 0.2 MPa, and increased rapidly for exposures above the apparent cavitation threshold (using the H2O2 production test) of about 0.4 MPa spatial peak pressure amplitude. In the DNA transfection tests, 20 micrograms/mL luciferase reporter plasmid was added to the suspension during exposure, and cells were assayed for proliferation ability and luciferase gene expression 2 days after exposure. Cell proliferation was greatly reduced above the cavitation threshold. Luciferase production was significant for 0.20-MPa exposure, and reached 0.33 ng per 10(6) cells at 0.8-MPa exposure. The luciferase production was great for cells exposed in medium supplemented with serum than for cells exposed in serum-free medium. Cells harvested for exposure either in the log phase or in the stationary phase of culture gave similar proliferation and transfection results. The effects essentially disappeared when the Albunex was omitted from the suspension and the tube was not rotated. Thus, sonoporation by ultrasonic cavitation in the rotating tube system yields plasmid transfection with subsequent transient gene expression.
Article
Application of ultrasound transiently permeabilizes cell membranes and offers a nonchemical, nonviral, and noninvasive method for cellular drug delivery. Although the ability of ultrasound to increase transmembrane transport has been well demonstrated, a systematic dependence of transport on ultrasound parameters is not known. This study examined cell viability and cellular uptake of calcein using 3T3 mouse cell suspension as a model system. Cells were exposed to varying acoustic energy doses at four different frequencies in the low frequency regime (20-100 kHz). At all frequencies, cell viability decreased with increasing acoustic energy dose, while the fraction of cells exhibiting uptake of calcein showed a maximum at an intermediate energy dose. Acoustic spectra under various ultrasound conditions were also collected and assessed for the magnitude of broadband noise and subharmonic peaks. While the cell viability and transport data did not show any correlation with subharmonic (f/2) emission, they correlated with the broadband noise, suggesting a dominant contribution of transient cavitation. A theoretical model was developed to relate reversible and irreversible membrane permeabilization to the number of transient cavitation events. The model showed that nearly every stage of transient cavitation, including bubble expansion, collapse, and subsequent shock waves may contribute to membrane permeabilization. For each mechanism, the volume around the bubble within which bubbles induce reversible and irreversible membrane permeabilization was determined. Predictions of the model are consistent with experimental data.
Article
We synthesized a MCM-41-type mesoporous silica nanosphere (MSN)-based gene transfection system, where second generation (G2) polyamidoamines (PAMAMs) were covalently attached to the surface of MSN. The G2-PAMAM-capped MSN material (G2-MSN) was used to complex with a plasmid DNA (pEGFP-C1) that encodes for an enhanced green fluorescence protein. The gene transfection efficacy, uptake mechanism, and biocompatibility of the G2-MSN system with various cell types, such as neural glia (astrocytes), human cervical cancer (HeLa), and Chinese hamster ovarian (CHO) cells, were investigated. The mesoporous structure of the MSN material allows membrane-impermeable molecules, such as pharmaceutical drugs and fluorescent dyes, to be encapsulated inside the MSN channels. The system renders the possibility to serve as a universal transmembrane carrier for intracellular drug delivery and imaging applications.
Article
One factor critical to successful gene therapy is the development of efficient delivery systems. Although advances in gene transfer technology, including viral and non-viral vectors, have been made, an ideal vector system has not yet been constructed. This review describes the basic principles behind various physical methods for gene transfer and assesses the advantages and performance of such approaches, compared to other transfection systems. In particular, the kinetics and efficiency of gene delivery, the toxicity, in vivo feasibility, and targeting ability of different physical methodologies are discussed and evaluated.
Article
The lack of safe and efficient gene-delivery methods is a limiting obstacle to human gene therapy. Synthetic gene-delivery agents, although safer than recombinant viruses, generally do not possess the required efficacy. In recent years, a variety of effective polymers have been designed specifically for gene delivery, and much has been learned about their structure-function relationships. With the growing understanding of polymer gene-delivery mechanisms and continued efforts of creative polymer chemists, it is likely that polymer-based gene-delivery systems will become an important tool for human gene therapy.
Article
Dendrimers have unique molecular architectures and properties that make them attractive materials for the development of nanomedicines. Key properties such as defined architecture and a high ratio of multivalent surface moieties to molecular volume also make these nanoscaled materials highly interesting for the development of synthetic (non-viral) vectors for therapeutic nucleic acids. Rational development of such vectors requires the link to be made between dendrimer structure and the morphology and physicochemistry of the respective nucleic acid complexes and, furthermore, to the biological performance of these systems at the cellular and systemic level. The review focuses on the current understanding of the role of dendrimers in those aspects of synthetic vector development. Dendrimer-based transfection agents have become routine tools for many molecular and cell biologists but therapeutic delivery of nucleic acids remains a challenge.
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.
Article
It has been proven, that the cellular uptake of drugs and genes is increased, when the region of interest is under ultrasound insonification, and even more when a contrast agent is present. This increased uptake has been attributed to the formation of transient porosities in the cell membrane, which are big enough for the transport of drugs into the cell (sonoporation). Owing to this technique, new ultrasound contrast agents that incorporate a therapeutic compound have become of interest. Combining ultrasound contrast agents with therapeutic substances, such a chemotherapeutics and virus vectors, may lead to a simple and economic method to instantly cure upon diagnosis, using conventional ultrasound scanners. There are two hypotheses for explaining the sonoporation phenomenon, the first being microbubble oscillations near a cell membrane, the second being microbubble jetting through the cell membrane. Based on modeling, high-speed photography, and recent cellular uptake measurements, it is concluded that microbubble jetting behavior is less likely to be the dominant sonoporation mechanism. Ultrasound-directed drug delivery using microbubbles is a promising method that has great potential in the treatment of malignant disorders.