Effects of microbubbles on ultrasound-mediated gene transfer in human prostate cancer PC3 cells: Comparison among Levovist, YM454, and MRX-815H
Akihiko WatanabeRemon OtakeTetsuo NozakiAkihiro MoriiRyohei OgawaShinichi FujimotoShinobu NakamuraHideki FuseTakashi Kondo
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Sonication
Gene transfer
Ultrasound microbubbles are a type of contrast agent that has acoustic characteristics that can be used in ultrasonic contrast imaging.The sensitivity and specificity of microbubbles in ultrasound would improve echocardiography and molecular imaging diagnosis.This article reviews the use of ultrasound microbubbles in the diagnosis of cardiovascular diseases.
Ultrasound imaging
Diagnostic ultrasound
Contrast-enhanced ultrasound
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Microbubbles are widely used as ultrasound contrast agents owing to their excellent echoing characteristics under ultrasound radiation. However, their short sonographic duration and wide size distribution still hinder their application. Herein, we present a hard-template approach to produce perfluoropropane-loaded cerasomal microbubbles (PLCMs) with uniform size and long sonographic duration. The preparation of PLCMs includes deposition of Si-lipids onto functionalized CaCO3 microspheres, removal of their CaCO3 cores and mild infusion of perfluoropropane. In vitro and in vivo experiments showed that PLCMs had excellent echoing characteristics under different ultrasound conditions. More importantly, PLCMs could be imaged for much longer than SonoVue (commercially used microbubbles) under the same ultrasound parameters and concentrations. Our results demonstrated that PLCMs have great potential for use as a novel contrast agent in ultrasound imaging.
Contrast-enhanced ultrasound
Ultrasound imaging
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Ultrasound contrast agents consist of gas-filled coated microbubbles that oscillate upon ultrasound insonification. Their characteristic oscillatory response provides contrast enhancement for imaging and has the potential to locally enhance drug delivery. Since microbubble response depends on the local acoustic pressure, an ultrasound compatible chamber is needed to study their behavior and the underlying drug delivery pathways. In this study, we determined the amplitude of the acoustic pressure in the CLINIcell, an optically transparent chamber suitable for cell culture. The pressure field was characterized based on microbubble response recorded using the Brandaris 128 ultrahigh-speed camera and an iterative processing method. The results were compared to a control experiment performed in an OptiCell, which is conventionally used in microbubble studies. Microbubbles in the CLINIcell responded in a controlled manner, comparable to those in the OptiCell. For frequencies from 1 to 4 MHz, the mean pressure amplitude was -5.4 dB with respect to the externally applied field. The predictable ultrasound pressure demonstrates the potential of the CLINIcell as an optical, ultrasound, and cell culture compatible device to study microbubble oscillation behavior and ultrasound-mediated drug delivery.
Oscillation (cell signaling)
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It has been shown that B-mode ultrasound can be useful for the real-time visualization of high-intensity focused ultrasound (HIFU) treatment. The aim of this study is to demonstrate the real-time ultrasound observation of functional changes when a vessel is exposed to pulsed-HIFU in the presence of preformed microbubbles. Using in vivo experiments, 12 male Sprague-Dawley rats were sonicated by 1-MHz pulsed-HIFU in the presence of ultrasound contrast agent (UCA) at four doses (0, 150, 300, and 450 μL/kg). The microbubbles passing through the aorta can be discerned with B-mode imaging. The mean peak systolic velocity (PSV) of the blood flow, as measured by Doppler ultrasound imaging, increased in arteries when the low-dose UCA groups (0 and 150 μL/kg) were examined after pulsed-HIFU at 45 W, but decreased when the high-dose UCA groups (300 and 450 μL/ kg) were examined. Additionally, the normalized pulsatility index (PI) changes increased with the injected dose of UCA. The interactions between ultrasound and the microbubbles can be seen to change the tissue permeability of the drug. Thus, monitoring of PSV or PI might be useful as an online method to ensure the correct sonicated position and to indicate when drug delivery has occurred.
Mechanical index
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Objective:The purpose of this study is to prepare streptoavidin(SA) conjugated anionic lipid microbubbles and to assess the physiochemical properties. Methods: The first step was to direct binding of indocarbocyanin(Cy3) labeled SA to microbubbles by mechanical vibration, sonication, surface bind after mechanical vibration or sonication which were divided into four groups. Half of all resulted suspensions were used for repeated washings, trying to remove any free Cy3-SA using floatation method. Then, the size, shape, bubble concentration and fluorescent intensity were measured and analyzed under light and fluorescent microscopy. The binding rate of microbubbles with Cy3-SA was calculated using flow cytometry. Results: Compared with mechanical vibration groups, anionic lipid microbubbles with Cy3-SA in sonication groups appeared to be slightly larger in size. The microbubbles with Cy3-SA delaminated quicker than the control group. All Cy3-SA bubbles could give a bright red fluorescence except the control group. Though microbubbles concentration dropped with washings, it was still fluorescent visible and no significant binding rates differences were found in different preparation methods(P0.05), it was above 98%. Conclusions: Streptoavidin can be conjugated with lipid microbubbles through mechanical vibration or sonication during preparation procedure. The conjugation of MB and SA were firmly high.
Sonication
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Ultrasound in combination with microbubbles (sonoporation) has recently acquired much attention in the field of gene delivery. The mechanism by which ultrasound mediates cellular delivery has been ascribed as cavitation. Cavitation is the alternate growing and shrinking of microbubbles as a result of the high and low pressure waves of the ultrasound. Eventually, these cavitating microbubbles can also implode due to these high pressure waves.The cavitation and especially the implosion of microbubbles generate local shock waves and microjets that can temporally perforate the cell membrane. However, a major limitation of the currently available microbubbles is that they have a short lifetime, and neither bind or protect the therapeutic DNA against nuclease. Consequently, the aim of this work was to develop ultrasound responsive microbubbles which can bind and protect DNA against nucleases. We developed new microbubbles by coating classic albumin microbubbles with a cationic charged polymer via the layer-by-layer technique. Albumin microbubbles were prepared by sonicating a dextrose- albumin solution with perfluorobutane gas. Poly(allylamine hydrochloride) (PAH) coated microbubbles were prepared starting from the microbubbles above using the Layer-by-Layer technique. About 90% of the uncoated and coated microbubbles had a size between 1 and 5 μm. Coating of the microbubbles with PAH, turned the surface charge positive. The albumin shell (green) of the uncoated microbubbles and the PAH coat (red) of the coated microbubbles was subsequently visualized using CSLM (figure 1A and 1B ). The appearance of a red colored ring around the microbubbles further proved that the microbubbles are indeed coated with PAH. Incubation of unlabeled, uncoated microbubbles with green labeled pDNA resulted in a rather homogeneous distribution of the fluorescence (Figure 1C). However, an accumulation of the pDNA around the microbubbles occurred when the unlabeled PAH coated microbubbles were incubated wih the green labeled pDNA (Figure 1D). Similar conclusions could be drawn from zeta potential measurements. The maximum loading capacity of the PAH coated microbubbles was estimated around 0.1 pg/ microbubble. A 5-fold increase of the half-life of the microbubbles was obtained after coating them with PAH. The ability of the microbubbles to protect pDNA against nuclease cleavage was tested using gel electrophoresis. The uncoated microbubbles were not able to protect pDNA from degradation by DNase I. Oppositely the PAH coated microbubbles were able to prevent degradation.
Sonoporation
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Ultrasound contrast agents are microbubbles composed of a thin lipid or albumin shell filled with air or a high molecular weight gas. These microbubbles are used for contrast-enhanced ultrasound (CEU) assessment of organ perfusion. In regions of inflammation, microbubbles are phagocytosed intact by activated neutrophils adherent to the venular wall. The authors hypothesized that microbubbles remain acoustically active following phagocytosis. Accordingly, they assessed the physical responses of both phagocytosed and free microbubbles by direct microscopic observation during delivery of repetitive single pulses of ultrasound at various acoustic pressures. Insonation results in oscillation in the bubbles volume. Microbubbles were optically recorded during insonation with a high-speed imaging system and diameter-time curves were analyzed to determine the effect of phagocytosis. Phagocytosed microbubbles retained their acoustic activity, although the intracellular environment increased viscoelastic damping experienced by microbubbles. With a pulse of high acoustic intensity (>1 MPa), phagocytosed microbubbles expanded up to 500% of their initial radii, which occasionally resulted in neutrophil rupture. Primary radiation force displaced phagocytosed microbubbles a distance of 100 microns with an acoustic pressure of -240 kPa and a pulse repetition frequency of 10 kHz, thus providing further evidence of acoustic activity. The authors conclude that phagocytosed microbubbles exhibit viscoelastic damping and yet are susceptible to acoustic destruction. They can generate non-linear echoes on the same order of magnitude as free microbubbles. These results indicate that CEU may be used to identify and assess regions of inflammation by detecting acoustic signals from microbubbles that are phagocytosed by activated neutrophils. In addition, the rapid expansion of a microbubble at high acoustic pressure may present a means to rupture a neutrophil or drug capsule at a specific site, resulting in delivery of a drug.
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Air-filled lysozyme microbubbles can be synthesized in an aqueous medium by emulsification followed by the cross-linking of protein molecules under high-intensity ultrasound. Here, we report on the tailoring of the properties of the ultrasonically synthesised microbubbles using new procedures. The efficiency of formation, size, size distribution and morphology of the microbubbles were controlled by manipulating the experimental conditions, namely, the sonication power and the length of sonication. An increase in the sonication time and power led to the formation of larger microbubbles with a broader size distribution. The microbubble shell thickness was found to decrease with an increase in the sonication power and time. Furthermore, a pulsed sonoluminescence technique was used to study the strength and stability of the microbubbles. The experimental results have shown that the effects of sonication time and power on the properties of the microbubbles are quite complex. A simple graphical matrix has been derived to obtain stable microbubbles with a narrow size distribution.
Sonication
Sonochemistry
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Targeted drug delivery
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Microbubbles are small spherical gas filled bubbles in the size range of 1-10 microns. Their external coat is made of polymers or phospholipids. In combination with ultrasound, they have been explored as contrast agents for ultrasound and also carriers for drug and gene delivery. In response to ultrasound of lower mechanical index, microbubbles oscillate and vibrate to give a distinct signal in ultrasound imaging. At a higher mechanical index, these microbubbles rupture and break to deliver the drugs or genes enclosed in them or attached to their surface. The behaviour of microbubbles in response to ultrasound is a characteristic of the properties of microbubbles like its shell composition, shell thickness and the density and compressibility of the enclosed gas. Microbubbles have various applications in diagnostic imaging like echocardiography, and imaging of cancer cells, inflammed cells etc. They are also used as a medium for drug and gene delivery. Microbubbles can be further modified by binding specific ligands to their surface which specifically attach to certain cells so that selective action of the microbubbles can be seen at that location of cells. Keywords: Acoustic impedance, Contrast agent, Drug delivery, Ligands, Microbubbles, Targeting, Ultrasound.
Mechanical index
Ultrasound imaging
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