Ten seconds into the ultrasound sonication, allowing five control pulses to be emitted, SonoVue® microbubbles (Bracco, Milan, Italy) were administered intravenously through a 30 G catheter over a 30 s time period (volume: 100 µl, concentration: 5 µl/g). The microbubbles were activated following manufacturers' instructions and were used within 6 h from activation. One minute from the start of the sonication, Gd(rhodamine-pip-DO3A) (molecular weight: 1 kDa, concentration: 5.6 mg/ml; n = 10) or lysine-fixable Texas Red® 3 kDa dextran (concentration: 5 mg/ml; Life Technologies, Paisley, UK; n = 6) was also injected. The probes were diluted in 100 µl phosphate-buffered saline (PBS) and were not expected to cross an intact BBB due to their size being over the 400 Da threshold 47 (link). Of the ten mice where Gd(rhodamine-pip-DO3A) was delivered, three brains were imaged with MRI as well as fluorescence microscopy.
Sonovue microbubbles
Manufactured by Bracco
Sourced in Italy, Switzerland
SonoVue microbubbles are a contrast agent used in diagnostic ultrasound imaging. The microbubbles are designed to enhance the visibility of blood flow and blood-containing structures during ultrasound examinations.
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Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (5 mentions)
Lovastatin, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Avantor (1 mentions)
Nocodazole, by Merck Group (1 mentions)
L mimosine, by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Ivis lumina xr system, by PerkinElmer (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Mir 125b 5p mimic, by GenePharma (1 mentions)
14 protocols using sonovue microbubbles
1
Microbubble-assisted BBB Permeability Imaging
2
Ultrasound Contrast Imaging of Tumors
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We injected 2.4 ml SonoVue microbubbles (Bracco company) into the patients’ elbow superficial vein, then 5.0 ml normal saline was injected. The targeted longitudinal tumor section was observed for more than 3 min. For patients with multiple target lesions, if the lesions could not be simultaneously displayed in the same section, we waited 6–10 min after angiography until the contrast agent completely disappeared before performing the next lesion examination.
3
Cell Synchronization and Sonoporation Techniques
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Gibco's RPMI medium 1640 (Carlsbad, CA, USA) with fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), penicillin/streptomycin (Gibco Inc., Carlsbad, CA, USA), Trypsin-EDTA (Gibco Inc., Carlsbad, CA, USA), phosphate buffer solution (PBS; Gibco Inc., Carlsbad, CA, USA) and dimethyl sulfoxide (DMSO; Amresco Inc., Solon, OH, USA) were used for cell culture. Lovastatin (M2147, Sigma-Aldrich, St. Louis, MO, USA), nocodazole (M1404, Sigma-Aldrich, St. Louis, MO, USA) and L-mimosine (M0253, Sigma-Aldrich, St. Louis, MO, USA) were used to synchronize cells in different cycle phases. Propidium Iodide (PI; P4170, Sigma-Aldrich, St. Louis, MO, USA) was used to indicate the variation in the cell membrane permeability. SonoVue® microbubbles (Bracco diagnostics Inc., Geneva, Switzerland) with a mean radius of ~1.5 μm were used as sonoporation agents. According to the manufacturer's instruction, immediately before the experiments, the vial of SonoVue® was vented with a sterile 18-gauge needle, followed by the addition of 5-ml PBS. Finally, ultrasound contrast agent (UCA) microbubbles with a concentration of 2-5×108 microbubbles/mL were evenly distributed by inversion agitation of the vial.
4
Preparation of SonoVue Microbubble Suspension
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SonoVue microbubbles (Bracco, Italy) consist of phospholipid shells filled with sulfur hexafluoride and have a diameter of 2–7 µm. According to the manufacturer's specifications, the microbubbles were freshly reconstituted in 5 mL of physiological saline solution to form a suspension with a concentration of 2–5 × 108/mL (Correas et al., 2001 (link)). The reconstituted SonoVue microbubbles were diluted in phosphate buffer saline (PBS) to a concentration of 20% (v/v).
5
Ultrasound Imaging of Tumor Perfusion
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All animal studies were approved by the Animal Experimentation Ethics Committee of Huazhong University of Science and Technology, and carried out in compliance with guidelines approved by the Science and Technology Department of Hubei Province.
BALB/c nude mice (male, aged 4 weeks, 18–20 g) were used in this study (Animals Center at Tongji Medical College). C6 cells (2×106 cells/mouse in 100 µL PBS) were subcutaneously injected into the right lower back of the mice. When the tumor volume reached 350–400 mm3, 100 µL of NBs were injected intra-tumorally. A probe (10 MHz) was placed at the tumor region to obtain images at the time points of 0 seconds, 30 seconds, 10 minutes, and 120 minutes. SonoVue® microbubbles (Bracco Imaging SpA, Milan, Italy), a commercial ultrasonic contrast agent, were used as control.
BALB/c nude mice (male, aged 4 weeks, 18–20 g) were used in this study (Animals Center at Tongji Medical College). C6 cells (2×106 cells/mouse in 100 µL PBS) were subcutaneously injected into the right lower back of the mice. When the tumor volume reached 350–400 mm3, 100 µL of NBs were injected intra-tumorally. A probe (10 MHz) was placed at the tumor region to obtain images at the time points of 0 seconds, 30 seconds, 10 minutes, and 120 minutes. SonoVue® microbubbles (Bracco Imaging SpA, Milan, Italy), a commercial ultrasonic contrast agent, were used as control.
6
Muscle Perfusion Measurement using CEUS
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CEUS permits the measurement MBF and its components, microvascular blood volume (MBV) and microvascular flow velocity (MFV) and was described in detail previously [40 (link)]. In brief, Sonovue microbubbles (Bracco, Milan, Italy) infused via an antecubital fossa vein were detected via ultrasound. A linear probe was positioned on the m. tibialis anterior and on the m. vastus lateralis to detect intravascular microbubble concentration in the muscles. To disrupt microbubbles, intermittent high mechanical index “flashes” were used, with subsequent continuous low mechanical index recording measuring the rate of microbubble reappearance after each flash. Initially, Sonovue was infused at 2 mL/min for 1 min and then 1 mL/min for 3 min thereafter. In total, Sonovue was infused for 4 min. At 2.5 min, 3 30 s flash/replenishment recordings were made across the last 90 s of the protocol at each CEUS time point. After each flash, a 0.48 s window was used to adjust for noncontrast signal and for rapid filling of larger conduit vessels. The acoustic intensity of insonated tissue in the postflash period demonstrates a first-order exponential association function with a rate constant that is proportional to MFV and a plateau proportional to MBV. During CEUS measurements (<10 min), volunteers were asked to remain quiet and still.
7
Nanobubble-enhanced Tumor Imaging in Rabbits
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The anaesthetized rabbits were injected with 1 mL of the nanobubbles through the ear vein. The probe (10 MHz) was used to obtain the tumor imaging. Sonovue microbubbles (Bracco, Italy), a commercial ultrasonic contrast agent, are used as control. The rabbits were sacrificed after completion of the experiments, and perfused with saline followed by 4% paraformaldehyde. The liver was removed and the tumor was separated, both of these were studied using an IVIS Lumina XR system (Caliper Life Sciences, USA). The ex vivo fluorescent images of the organs were acquired and analyzed. Finally, the organs were treated with hematoxylineeosin (H & E) staining.
8
Stroke Treatment with BMSC and UTMD
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The rats were randomly assigned to either the sham-operated group or the MCAO model group. The sham-operated rats underwent surgical exposure of the arterial vessels without occluding the common carotid arteries. All rats in the sham-operated group were assigned a Longa score of 0. Twenty-four hours after performing the MCAO model, rats with a Longa Score of 2 were subsequently randomized into the following groups: ACI+PBS group, ACI+BMSC group, and ACI+BMSC+UTMD group. Phosphate-buffered saline (PBS) of 1 mL was injected intravenously via the tail vein in ACI+PBS group 24 h after MCAO induced. A total of 5 × 106 BMSC in 1 mL PBS were injected intravenously via the tail vein in ACI+BMSC group. In the ACI+BMSC+UTMD group, intravenous injection of SonoVue microbubbles (Bracco, Milan, Italy) at a dose of 0.1 mL/kg was performed via the tail vein, followed by acoustic stimulation. After the stimulation, 5 × 106 BMSC cells were injected intravenously.
9
Enhancing miR-378 Delivery via Ultrasound
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In order to explore the role of miR-378 and the efficiency of the combined method for cell transfection, the cells were grouped as Blank, miR-378 control, L group (HCC cells transfected with miR-378) and LUS groups (HCC cells treated with miR-378 mimic combined with ultrasonic irradiation and SonoVue microbubbles). Briefly, HuH-7, Hep3B and SK-Hep1 cells at 1×106 cells/ml in the LUS or miR-378 control groups were plated in a 96-well plate and then respectively transfected with 100 nmol/l miR-378 mimic (Shanghai GenePharma Co., Ltd.), or miR-378 mimic control vector (Shanghai GenePharma Co., Ltd.) in a mixture with Lipofectamine® 3000 (L3000015; Thermo Fisher Scientific, Inc.) and 2.5 µg/µl SonoVue microbubbles (Bracco Suisse SA) under the irradiation of ultrasonic transfer apparatus via ultrasound couplant (Anhui Deepblue Medical Technology Co., Ltd.) at the parameters of 0.5 W/cm2 for 30 sec. The cells in the L group were transfected with miR-378 mimic using Lipofectamine 3000 only, while those in the Blank group were treated with medium only. All the cells were cultured for another 72 h after the transfection (19 (link)). The sequence of miR-378 mimic was 5′-AGGCUCUGACUCCAGGUCC-3′; The sequence of miR-378 mimic control was 5′-UUCUCCGAACGUGUCACGUTT-3′.
10
Reconstitution and Fabrication of Microbubbles
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SonoVue® microbubbles (Bracco Spa, Milan, Italy) consist of a phospholipid shell encapsulating a sulfur hexafluoride gas core. Before the experiment, the shells are reconstituted through a mixture of the lyophilisate with 5 ml physiological saline solution, to form a suspension that contains ~ 2−5 × 108 microbubbles per milliliter with radius ranging from 2 to 7 μm.
We also used lipid microbubbles encapsulating C4F10 with a more narrow size distribution that the one we produced in a flow-focusing device. The fabrication process of these DSPC/DPPE-PEG5000 microbubbles is summarized in ref. 21 (link), following protocols detailed in refs. 43 (link),44 (link).
We also used lipid microbubbles encapsulating C4F10 with a more narrow size distribution that the one we produced in a flow-focusing device. The fabrication process of these DSPC/DPPE-PEG5000 microbubbles is summarized in ref. 21 (link), following protocols detailed in refs. 43 (link),44 (link).
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