Site-specific sonoporation of human melanoma cells at the cellular level using high lateral-resolution ultrasonic micro-transducer arrays.
ABSTRACT: We developed a new instrumental method by which human melanoma cells (LU1205) are sonoporated via radiation pressures exerted by highly-confined ultrasonic waves produced by high lateral-resolution ultrasonic micro-transducer arrays (UMTAs). The method enables cellular-level site-specific sonoporation within the cell monolayer due to UMTAs and can be applicable in the delivery of drugs and gene products in cellular assays. In this method, cells are seeded on the biochip that employs UMTAs for high spatial resolution and specificity. UMTAs are driven by 30-MHz sinusoidal signals and the resulting radiation pressures induce sonoporation in the targeted cells. The sonoporation degree and the effective lateral resolution of UMTAs are determined by performing fluorescent microscopy and analysis of carboxylic-acid-derivatized CdSe/ZnS quantum dots passively transported into the cells. Models representing the transducer-generated ultrasound radiation pressure, the ultrasound-inflicted cell membrane wound, and the transmembrane transport through the wound are developed to determine the ultrasound-pressure-dependent wound size and enhanced cellular uptake of nanoparticles. Model-based calculations show that the effective wound size and cellular uptake of nanoparticles increase linearly with increasing ultrasound pressure (i.e., at applied radiation pressures of 0.21, 0.29, and 0.40 MPa, the ultrasound-induced initial effective wound radii are 150, 460, and 650 nm, respectively, and the post-sonoporation intracellular quantum-dot concentrations are 7.8, 22.8, and 29.9 nM, respectively) and the threshold pressure required to induce sonoporation in LU1205 cells is ?0.12 MPa.
Project description:The flow fields generated by the acoustic behavior of microbubbles can significantly increase cell permeability. This facilitates the cellular uptake of external molecules in a process known as ultrasound-mediated drug delivery. To promote its clinical translation, this study investigated the relationships among the ultrasound parameters, acoustic behavior of microbubbles, flow fields, and delivery results. SonoVue microbubbles were activated by 1 MHz pulsed ultrasound with 100 Hz pulse repetition frequency, 1:5 duty cycle, and 0.20/0.35/0.70 MPa peak rarefactional pressure. Micro-particle image velocimetry was used to detect the microbubble behavior and the resulting flow fields. Then HeLa human cervical cancer cells were treated with the same conditions for 2, 4, 10, 30, and 60 s, respectively. Fluorescein isothiocyanate and propidium iodide were used to quantitate the rates of sonoporated cells with a flow cytometer. The results indicate that (1) microbubbles exhibited different behavior in ultrasound fields of different peak rarefactional pressures. At peak rarefactional pressures of 0.20 and 0.35 MPa, the dispersed microbubbles clumped together into clusters, and the clusters showed no apparent movement. At a peak rarefactional pressure of 0.70 MPa, the microbubbles were partially broken, and the remainders underwent clustering and coalescence to form bubble clusters that exhibited translational oscillation. (2) The flow fields were unsteady before the unification of the microbubbles. After that, the flow fields showed a clear pattern. (3)The delivery efficiency improved with the shear stress of the flow fields increased. Before the formation of the microbubble/bubble cluster, the maximum shear stresses of the 0.20, 0.35, and 0.70 MPa groups were 56.0, 87.5 and 406.4 mPa, respectively, and the rates of the reversibly sonoporated cells were 2.4% ± 0.4%, 5.5% ± 1.3%, and 16.6% ± 0.2%. After the cluster formation, the maximum shear stresses of the three groups were 9.1, 8.7, and 71.7 mPa, respectively. The former two could not mediate sonoporation, whereas the last one could. These findings demonstrate the critical role of flow fields in ultrasound-mediated drug delivery and contribute to its clinical applications.
Project description: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.
Project description:Low-intensity ultrasound-microbubble (LIUS-MB) treatment is a promising antivascular therapy for tumors. We sought to determine whether LIUS-MB treatment with an appropriate ultrasound pressure could achieve substantial and persistent cessation of tumor perfusion without having significant effects on normal tissue. Further, we investigated the mechanisms underlying this treatment. Murine S-180 sarcomas, thigh muscles, and skin tissue from 60 tumor-bearing mice were subjected to sham therapy, an ultrasound application combined with microbubbles in four different ultrasound pressures (0.5, 1.5, 3.0, 5.0 MPa), or ultrasound at 5.0 MPa alone. Subsequently, contrast-enhanced ultrasonic imaging and histological studies were performed. Tumor microvessels, tumor cell necrosis, apoptosis, tumor growth, and survival were evaluated in 85 mice after treatment with the selected ultrasound pressure. We found that twenty-four hours after LIUS-MB treatment at 3.0 MPa, blood perfusion and microvessel density of the tumor had substantially decreased by 84?±?8% and 84%, respectively (p?<?0.01). Similar reductions were not observed in the muscle or skin. Additionally, an extreme reduction in the number of immature vessels was observed in the tumor (reduced by 90%, p?<?0.01), while the decrease in mature vessels was not significant. Further, LIUS-MB treatment at 3.0 MPa promoted tumor cell necrosis and apoptosis, delayed tumor growth, and increased the survival rate of tumor-bearing mice (p?<?0.01). These findings indicate that LIUS-MB treatment with an appropriate ultrasound pressure could selectively and persistently reduce tumor perfusion by depleting the neovasculature. Therefore, LIUS-MB treatment offers great promise for clinical applications in antivascular therapy for solid tumors.
Project description:Sonoporation refers to the use of ultrasound and acoustic cavitation to temporarily enhance the permeability of cellular membranes so as to enhance the delivery efficiency of therapeutic agents into cells. Microbubble-based ultrasound contrast agents are often used to facilitate these cavitation effects. This study used nanodroplets to significantly enhance the effectiveness of sonoporation relative to using conventional microbubbles. Significant enhancements were demonstrated both in vitro and in vivo by using gold nanorods encapsulated in nanodroplets for implementing plasmonic photothermal therapy. Combined excitation by ultrasound and laser radiation is used to trigger the gold nanodroplets to induce a liquid-to-gas phase change, which induces cavitation effects that are three-to-fivefold stronger than when using conventional microbubbles. Enhanced cavitation also leads to significant enhancement of the sonoporation effects. Our in vivo results show that nanodroplet-vaporization-assisted sonoporation can increase the treatment temperature by more than 10 °C above that achieved by microbubble-based sonoporation.
Project description:We demonstrate a new optical approach to generate high-frequency (>15?MHz) and high-amplitude focused ultrasound, which can be used for non-invasive ultrasound therapy. A nano-composite film of carbon nanotubes (CNTs) and elastomeric polymer is formed on concave lenses, and used as an efficient optoacoustic source due to the high optical absorption of the CNTs and rapid heat transfer to the polymer upon excitation by pulsed laser irradiation. The CNT-coated lenses can generate unprecedented optoacoustic pressures of >50?MPa in peak positive on a tight focal spot of 75??m in lateral and 400??m in axial widths. This pressure amplitude is remarkably high in this frequency regime, producing pronounced shock effects and non-thermal pulsed cavitation at the focal zone. We demonstrate that the optoacoustic lens can be used for micro-scale ultrasonic fragmentation of solid materials and a single-cell surgery in terms of removing the cells from substrates and neighboring cells.
Project description:Due to their low cost, hand-held convenience, wide selection of bandwidths, and ultrasound imaging capability, linear ultrasonic transducer arrays have been widely studied for photoacoustic computed tomography (PACT). As linear-array PACT suffers from a limited view, full-view imaging requires either the transducer or the object to be rotated. So far, both the central frequencies and bandwidth of linear transducer arrays applied in full-view PACT are low, limiting the spatial resolutions of the reconstructed images. Here, we present a multiview high-frequency PACT imaging system implemented with a commercial 40-MHz central frequency linear transducer array. By rotating the object through multiple angles with respect to the linear transducer array, we acquired full-view photoacoustic pressure measurements. Further, to quantify the unipolar initial pressures and overcome the limitations of the single-view Hilbert transformation, we developed a multiview Hilbert transformation method. The in-plane spatial resolution of this full-view linear-array PACT was quantified to be isotropically 60???m within a 10×10??mm² field of view. The system was demonstrated by imaging both a leaf skeleton and a zebrafish in vivo.
Project description:Sonoporation uses ultrasound, with the aid of ultrasound contrast agents (UCAs), to enhance cell permeabilization, thereby allowing delivery of therapeutic compounds noninvasively into specific target cells. The objective of this study was to determine if a computational model describing shear stress on a cell membrane due to microstreaming would successfully reflect sonoporation activity with respect to the peak rarefactional pressure. The theoretical models were compared to the sonoporation results from Chinese hamster ovary cells using Definity(®) at 0.9, 3.15, and 5.6 MHz and were found to accurately describe the maximum sonoporation activity, the pressure where a decrease in sonoporation activity occurs, and relative differences between maximum activity and the activity after that decrease. Therefore, the model supports the experimental findings that shear stress on cell membranes secondary to oscillating UCAs results in sonoporation.
Project description:Hydrostatic pressure is one of the physical factors affecting cellular physiology. Hydrostatic pressure of a few hundred MPa decreases the viability of yeast cells, and pressure of a few tens MPa decreases the growth rate. To understand the effect of hydrostatic pressure, we employed yeast, Saccharomyces cerevisiae, DNA microarrays and analyzed genome-wide mRNA expression profiles under hydrostatic pressures. In this experiment, we selected a hydrostatic pressure of 40 MPa at 25 degrees C because the condition is not lethal for yeast cells but the growth was suppressed. Keywords: stress response Overall design: Series contains triplicate hybridization results from independent biological samples respectively. From 1st to 3th hybridization data have single scanned data, on the other hand, 4th data have high and low power scanned data.
Project description:Hydrostatic pressure is one of the physical factors affecting cellular physiology. Hydrostatic pressure of a few hundred MPa decreases the viability of yeast cells, and pressure of a few tens MPa decreases the growth rate. To understand the effect of hydrostatic pressure, we employed yeast, Saccharomyces cerevisiae, DNA microarrays and analyzed genome-wide mRNA expression profiles under hydrostatic pressures. In this experiment, we selected a hydrostatic pressure of 30 MPa at 25 degrees C because yeast cells are able to grow with this condition. Keywords: stress response Overall design: Series contains triplicate hybridization results from independent biological samples respectively. 1st and 2nd hybridization data have high and low power scanned data, respectively. On the other hand, 3rd hybridization has single scanned data.
Project description:Focused ultrasound has been shown to be effective at stimulating neurons in many animal models, both in vivo and ex vivo Ultrasonic neuromodulation is the only noninvasive method of stimulation that could reach deep in the brain with high spatial-temporal resolution, and thus has potential for use in clinical applications and basic studies of the nervous system. Understanding the physical mechanism by which energy in a high acoustic frequency wave is delivered to stimulate neurons will be important to optimize this technology. We imaged the isolated salamander retina of either sex during ultrasonic stimuli that drive ganglion cell activity and observed micron scale displacements, consistent with radiation force, the nonlinear delivery of momentum by a propagating wave. We recorded ganglion cell spiking activity and changed the acoustic carrier frequency across a broad range (0.5-43 MHz), finding that increased stimulation occurs at higher acoustic frequencies, ruling out cavitation as an alternative possible mechanism. A quantitative radiation force model can explain retinal responses and could potentially explain previous in vivo results in the mouse, suggesting a new hypothesis to be tested in vivo Finally, we found that neural activity was strongly modulated by the distance between the transducer and the electrode array showing the influence of standing waves on the response. We conclude that radiation force is the dominant physical mechanism underlying ultrasonic neurostimulation in the ex vivo retina and propose that the control of standing waves is a new potential method to modulate these effects.SIGNIFICANCE STATEMENT Ultrasonic neurostimulation is a promising noninvasive technology that has potential for both basic research and clinical applications. The mechanisms of ultrasonic neurostimulation are unknown, making it difficult to optimize in any given application. We studied the physical mechanism by which ultrasound is converted into an effective energy form to cause neurostimulation in the retina and find that ultrasound acts via radiation force leading to a mechanical displacement of tissue. We further show that standing waves have a strong modulatory effect on activity. Our quantitative model by which ultrasound generates radiation force and leads to neural activity will be important in optimizing ultrasonic neurostimulation across a wide range of applications.