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      Microbubble-Mediated Ultrasound Enhances the Lethal Effect of Gentamicin on Planktonic Escherichia coli

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          Abstract

          Previous research has found that low-intensity ultrasound enhanced the lethal effect of gentamicin on planktonic E. coli. We aimed to further investigate whether microbubble-mediated low-intensity ultrasound could further enhance the antimicrobial efficacy of gentamicin. The planktonic E. coli (ATCC 25922) was distributed to four different interventions: control (G CON), microbubble only (G MB), ultrasound only (G US), and microbubble-mediated ultrasound (G MUS). Ultrasound was applied with 100 mW/cm 2 (average intensity) and 46.5 KHz, which presented no bactericidal activity. After 12 h, plate counting was used to estimate the number of bacteria, and bacterial micromorphology was observed with transmission electron microscope. The results showed that the viable counts of E. coli in G MUS were decreased by 1.01 to 1.42 log 10 CFU/mL compared with G US ( P < 0.01). The minimal inhibitory concentration (MIC) of gentamicin against E. coli was 1  μ g/mL in the G MUS and G US groups, lower than that in the G CON and G MB groups (2  μ g/mL). Transmission electron microscopy (TEM) images exhibited more destruction and higher thickness of bacterial cell membranes in the G MUS than those in other groups. The reason might be the increased permeability of cell membranes for gentamicin caused by acoustic cavitation.

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          Most cited references32

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          Ultrasound and microbubble-targeted delivery of macromolecules is regulated by induction of endocytosis and pore formation.

          Contrast microbubbles in combination with ultrasound (US) are promising vehicles for local drug and gene delivery. However, the exact mechanisms behind intracellular delivery of therapeutic compounds remain to be resolved. We hypothesized that endocytosis and pore formation are involved during US and microbubble targeted delivery (UMTD) of therapeutic compounds. Therefore, primary endothelial cells were subjected to UMTD of fluorescent dextrans (4.4 to 500 kDa) using 1 MHz pulsed US with 0.22-MPa peak-negative pressure, during 30 seconds. Fluorescence microscopy showed homogeneous distribution of 4.4- and 70-kDa dextrans through the cytosol, and localization of 155- and 500-kDa dextrans in distinct vesicles after UMTD. After ATP depletion, reduced uptake of 4.4-kDa dextran and no uptake of 500-kDa dextran was observed after UMTD. Independently inhibiting clathrin- and caveolae-mediated endocytosis, as well as macropinocytosis significantly decreased intracellular delivery of 4.4- to 500-kDa dextrans. Furthermore, 3D fluorescence microscopy demonstrated dextran vesicles (500 kDa) to colocalize with caveolin-1 and especially clathrin. Finally, after UMTD of dextran (500 kDa) into rat femoral artery endothelium in vivo, dextran molecules were again localized in vesicles that partially colocalized with caveolin-1 and clathrin. Together, these data indicated uptake of molecules via endocytosis after UMTD. In addition to triggering endocytosis, UMTD also evoked transient pore formation, as demonstrated by the influx of calcium ions and cellular release of preloaded dextrans after US and microbubble exposure. In conclusion, these data demonstrate that endocytosis is a key mechanism in UMTD besides transient pore formation, with the contribution of endocytosis being dependent on molecular size.
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            Vibrating microbubbles poking individual cells: drug transfer into cells via sonoporation.

            Ultrasound contrast microbubbles have the ability to enhance endothelial cell permeability and thus may be used as a new way to deliver drugs. It facilitates the transfer of extracellular molecules into cells activated through ultrasound driven microbubbles. The present study is designed to correlate the relationship between microbubble induced cell deformation and enhanced cell membrane permeability. Propidium iodide (PI) was used as a membrane integrity probe. Using high-speed imaging of vibrating microbubbles against endothelial cells and imaging transport of PI into these cells showed a direct correlation between cell deformation and resulting cell membrane permeability. The membrane permeabilization lasted for a short period without affecting endothelial cells viability. We identified that microbubbles are crucial to enhance transient cell membrane permeability. Thus, permeability of individual cells is increased. The roles of ultrasound contrast microbubbles as the trigger for improved drug efficacy are discussed.
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              Induction of cell-membrane porosity by ultrasound.

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                Author and article information

                Journal
                Biomed Res Int
                Biomed Res Int
                BMRI
                BioMed Research International
                Hindawi Publishing Corporation
                2314-6133
                2314-6141
                2014
                15 May 2014
                : 2014
                : 142168
                Affiliations
                Department of Orthopaedic Surgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, No. 88 Jiefang Road, Hangzhou 310009, China
                Author notes

                Academic Editor: Konstantinos Anagnostakos

                Author information
                http://orcid.org/0000-0002-0595-9603
                Article
                10.1155/2014/142168
                4052079
                7c4dcc69-8999-4e57-af73-cf6d68b69264
                Copyright © 2014 Han-Xiao Zhu et al.

                This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 22 January 2014
                : 23 April 2014
                : 27 April 2014
                Funding
                Funded by: http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China
                Award ID: 81101377,
                Funded by: http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China
                Award ID: 81101345,
                Funded by: http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China
                Award ID: 81171687,
                Funded by: http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China
                Award ID: 81371954,
                Funded by: http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China
                Award ID: 81201414,
                Funded by: http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China
                Award ID: 81201416
                Funded by: Key Project of Zhejiang Provincial Department of Science and Technology
                Award ID: 2011C13033),
                Funded by: Zhejiang Provincial Natural Science Foundation of China
                Award ID: Y2100161,
                Funded by: Zhejiang Provincial Natural Science Foundation of China
                Award ID: Y2090283
                Funded by: Scientific Research Fund of Zhejiang Provincial Education Department
                Award ID: Y201018936
                Funded by: Health Department of Zhejiang Province
                Award ID: 2012RCA032
                Categories
                Research Article

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