Effects of Perfluorocarbon Gases on the Size and Stability Characteristics of Phospholipid-Coated Microbubbles: Osmotic Effect versus Interfacial Film Stabilization

Micrometer-sized bubbles coated with phospholipids are used as contrast agents for ultrasound imaging and have potential for oxygen, drug, and gene delivery and as therapeutic devices. An internal perfluorocarbon (<i>F</i>C) gas is generally used to stabilize them osmotically. We report here on the effects of three relatively heavy <i>F</i>Cs, perfluorohexane (<i>F</i>-hexane), perfluorodiglyme (<i>F</i>-diglyme ), and perfluorotriglyme (<i>F</i>-triglyme), on the size and stability characteristics of microbubbles coated with a soft shell of dimyristoylphosphatidylcholine (DMPC) and on the surface tension and compressibility of DMPC monolayers. Monomodal populations of small bubbles (∼1.3 ± 0.2 μm in radius, polydispersivity index ∼8%) were prepared by sonication, followed by centrifugal fractionation. The mean microbubble size, size distribution, and stability were determined by acoustical attenuation measurements, static light scattering, and optical microscopy. The half-lives of <i>F</i>-hexane- and <i>F</i>-diglyme-stabilized bubbles (149 ± 8 and 134 ± 3 min, respectively) were about 2 times longer than with the heavier <i>F</i>-triglyme (76 ± 7 min) and 4–5 times longer than with air (34 ± 3 min). Remarkably, the bubbles are smaller than the minimal size values calculated assuming that the bubbles are stabilized osmotically by the insoluble <i>F</i>C gases. Particularly striking is that bubbles 2 orders of magnitude smaller than the calculated collapse radius can be prepared with <i>F</i>-triglyme, while its very low vapor pressure prohibits any osmotic effect. The interface between an aqueous DMPC dispersion and air, or air (or N₂) saturated with the <i>F</i>Cs, was investigated by tensiometry and by Langmuir monolayer compressions. Remarkably, after 3 h, the tensions at the interface between an aqueous DMPC dispersion (0.5 mmol L^–1) and air were lowered from ∼50 ± 1 to ∼37 ± 1 mN m^–1 when <i>F</i>-hexane and <i>F</i>-diglyme were present and to ∼40 ± 1 mN m^–1 for <i>F</i>-triglyme. Also noteworthy, the adsorption kinetics of DMPC at the interface, as obtained by dynamic tensiometry, were accelerated up to 3-fold when the <i>F</i>C gases were present. The compression isotherms show that all these <i>F</i>C gases significantly increase the surface pressure (from ∼0 to ∼10 mN m^–1) at large molecular areas (70 Ų), implying their incorporation into the DMPC monolayer. All three <i>F</i>C gases increase the monolayer’s collapse pressures significantly (∼61 ± 2 mN m^–1) as compared to air (∼54 ± 2 mN m^–1), providing for interfacial tensions as low as ∼11 mN m^–1 (vs ∼18 mN m^–1 in their absence). The <i>F</i>C gases increase the compressibility of the DMPC monolayer by 20–50%. These results establish that, besides their osmotic effect, <i>F</i>C gases contribute to bubble stabilization by decreasing the DMPC interfacial tension, hence reducing the Laplace pressure. This contribution, although significant, still does not suffice to explain the large discrepancy observed between calculated and experimental bubble half-lives. The case of <i>F</i>-triglyme, which has no osmotic effect, indicates that its effects on the DMPC shell (increased collapse pressure, decreased interfacial tension, and increased compressibility) contribute to bubble stabilization. <i>F</i>-hexane and <i>F</i>-diglyme provided both the smallest mean bubble sizes and the longest bubble half-lives

A New Type of Methacrylate-Substituted Oxozirconium Clusters: [Zr3O(OR)5(OMc)5]2 and [Zr3O(OR)3(OMc)7]2

 The new clusters Zr6O2(OBu)10(OMc)10 and Zr6O2(OMe)4(OBu)2(OMc)14 were prepared from Zr(OBu)4 and methacrylic acid. For the preparation of Zr6O2(OMe)4(OBu)2(OMc)14, partial exchange of the butoxy groups is necessary. The clusters consist of two Zr3 subunits that dimerize via alkoxide bridges. The structures are of the same type, except that four terminal butoxy ligands in Zr6O2(OBu)10(OMc)10 are exchanged for chelating methacrylate ligands in Zr6O2(OMe)4(OBu)2(OMc)14

miR-194 targets the talin2 gene and downregulates talin2 protein levels.

<p>(<b>A</b>) Alignment of miR-194 with <i>talin2</i> (<i>TLN2</i>) 3′-UTRs. Complementary sequences of miR-194 and mammalian <i>talin2</i> 3′-UTRs are marked in Bold. The seed sequences of miR-194 are underlined. Has, human; Ptr, pan troglotydes; Mmu, mus musculus; Rno, rat; Ocu, rabbit; Eeu, hedgehog; Cfa, dog; Fca, cat; Eca, horse; Laf, elephant. The underlined seed nucleotides were deleted in the <i>talin2</i> 3′-UTR mutant reporter construct described in (B). (<b>B</b>) Effect of miR-194 expression on the luciferase activities of wild-type and mutated talin2 3′-UTR reporters. MDA-MB-361 cells were transiently transfected with a miR Control or miR-194 precursor for 36 hrs. Luciferase activity was determined using a dual luciferase assay. ** <i>p</i><0.01. (<b>C</b>) Effect of miR-194 expression on talin2 protein levels in SKBr3 cells. SKBr3 cells were transiently transfected with a miR-194 precursor or a control miRNA (miR CTRL) for 48 hrs. Total protein was prepared and subjected to Western blotting. (<b>D</b>) Effect of talin2 downregulation on cell migration in SKBr3 cells. SKBr3 cells were transiently transfected with two siTalin2 (siTLN2 #7 and #8) or its control siRNA (siCTRL) for 48 hrs and then motility was measured overnight in a Transwell assay. <b>*</b> p<0.05 compared to siCTRL. (<b>E</b>) Effect of talin2 downregulation on cell invasion. SKBr3 cells were transiently transfected with two siTalin2 (siTLN2 #7 and #8) or its control siRNA (siCTRL) for 48 hrs and then invasion assay was performed. <b>*</b> p<0.05 compared to siCTRL. (<b>F</b>) Validation of talin2 siRNA efficacy. SKBr3 cells were transiently transfected with two siTalin2 (siTLN2 #7 and #8) or its control siRNA (siCTRL) or the transfection reagent only (mock) for 48 hrs and total protein was prepared. Western blotting was performed with a talin2 antibody.</p

miRNAs affected by trastuzumab in SKBr3 cells.

<p>miRNAs affected by trastuzumab in SKBr3 cells.</p

miR-194 inhibitor stimulates cell migration and blocks trastuzumab-inhibited cell migration.

<p>(<b>A</b>) Effect of miR-194 inhibitor on cell migration in SKBr3 cells. SKBr3 cells were transiently transfected with a miR-194 inhibitor or its negative control (miR inhibitor CTRL) for 48 hrs before measurement of migration overnight. <b>*</b> p<0.05 compared to the negative control. (<b>B</b>) Validation of efficacy of miR-194 inhibition. SKBr3 cells were transiently transfected with a miR-194 inhibitor or its negative control (miR inhibitor CTRL) for 48 hrs. Total RNA was prepared and miR-194 measured by QRT-PCR. <b>*</b> p<0.05 compared to miR inhibitor control. (<b>C</b>) Effect of miR-194 inhibitor on trastuzumab-inhibited cell migration. SKBr3 cells were transiently transfected with a miR-194 inhibitor or its negative control (miR inhibitor CTRL) for 16 hrs, treated with trastuzumab (Tras) or control hIgG (10 µg/ml) for 36 hrs, and motility was then measured for overnight in Transwell assays. <b>*</b> p<0.05.</p

List of prognostic miRNAs for overall survival. Hazard ratios with confidence intervals, Wald <i>P</i>-values and Bonferroni adjusted <i>P</i>-values are given.

<p>List of prognostic miRNAs for overall survival. Hazard ratios with confidence intervals, Wald <i>P</i>-values and Bonferroni adjusted <i>P</i>-values are given.</p

miRNAs affected by trastuzumab in BT474 cells.

<p>miRNAs affected by trastuzumab in BT474 cells.</p

Trastuzumab treatment downregulates talin2 protein expression and inhibits breast cancer cell migration.

<p>(<b>A</b>) Effect of trastuzumab (Tras) on cell migration in a scratch assay. BT474 and SKBr3 cells were seeded in 6-well plates. After cells had grown to confluence, a scratch was made in the monolayer. Cells were treated with trastuzumab (Tras) or control hIgG (10 µg/ml) for 72 hrs. Images were recorded at 72 hrs at 40× enlargement. (<b>B</b>) Effect of trastuzumab on cell migration over a shorter interval (16 h) in a scratch assay. SKBr3 cells were seeded in 6-well culture plates and cultured overnight to achieve a cell density of full confluence. A scratch was made in the monolayer. Cells were then treated with trastuzumab (Tras) or control hIgG (10 µg/ml) plus epidermal growth factor (EGF, 20 ng/ml, sigma) for 16 hrs. Images were recorded at the end of 16 hrs of EGF stimulation at 40× enlargement. (<b>C</b>) Effect of trastuzumab (Tras) on cytoskeletal vinculin distribution after IF staining. SKBr3 cells were treated with trastuzumab (Tras) or control hIgG (10 µg/ml) for 48 hrs, and then subjected to IF staining as described in Methods. (<b>D</b>) Effect of trastuzumab on talin2 protein in BT474 cells. BT474 cells were treated in vitro with different concentrations of trastuzumab for 48 hrs. Total protein was prepared and subjected to Western blotting. (<b>E</b>) Quantitation of talin2 expression. The talin2 bands on immunoblots from three different experiments including the one shown in (<b>D</b>) were digitized, normalized to the levels of GAPDH, and expressed as mean levels (error bars correspond stand deviation). The talin2 expression at trastuzumab 0 concentration was set at 1. (<b>F</b>) Effect of trastuzumab on talin2 protein in SKBr3 cells. SKBr3 cells were treated with trastuzumab (Tras) or control hIgG (10 µg/ml) for 48 hrs. Total protein was prepared and subjected to Western blotting. (<b>G</b>) Effect of trastuzumab on talin2 protein in BT474 xenografts. BT474 xenografts in nude mice were treated with trastuzumab (Tras) or hIgG 1 mg/kg intraperitoneally twice a week and for 3 weeks. Total cell lysates were prepared and subjected to Western blotting.</p

Concordance, Univariable and Multivariable Cox analyses of overall survival and progression-free survival in 138 patients, with metastatic colorectal cancer treated with 3^rd line cetuximab and irinotecan according to a miRNAs, gender, age, performance status and <i>KRAS</i> and, <i>PI3KCA</i> mutation status. miRNAs are tested as continuous variables in interquartile ranges units.

<p>Concordance, Univariable and Multivariable Cox analyses of overall survival and progression-free survival in 138 patients, with metastatic colorectal cancer treated with 3^rd line cetuximab and irinotecan according to a miRNAs, gender, age, performance status and <i>KRAS</i> and, <i>PI3KCA</i> mutation status. miRNAs are tested as continuous variables in interquartile ranges units.</p

Increased miR-194 expression inhibits breast cancer cell migration and invasion.

<p>(<b>A</b>) miR-194 expression in the stable clones of BT474 cells. BT474 cells were stably transfected with an empty pEGFP-C1 vector or pEGFP-miR-194 vector under the selection of G418. Two control clones #17 and #19 that contain empty vector and two miR-194-expressing clones #22 and #23 were established and subjected for QRT-PCR analysis. Hsa-miR-194 was purchased from ABI (Assay ID 000493). (<b>B</b>) Cell viability assay of BT474 stable cells that express miR-194 or its control vector. BT474 cells were stably transfected with empty pEGFP-C1 vector or pEGFP-miR194 construct under the selection of G418. Two control clones #17 and #19 that contain empty vector and two miR-194-expressing clones #22 and #23 were chosen to measure viability of crystal violet-stained cells on day 1, day 3 and day 5. (<b>C</b>) Effect of miR-194 precursor on cell migration in SKBr3 cells. SKBr3 cells were transiently transfected with a miR-194 precursor or a control miRNA (miR CTRL) for 48 hrs and motility was measured overnight in a Transwell assay. (<b>D</b>) Quantitation of the SKBr3 cell migration as shown in (C). <b>*</b> p<0.05 compared to miR control. (<b>E</b>) Effect of miR-194 precursor on cell invasion in SKBr3 cells. SKBr3 cells were transiently transfected with a miR-194 precursor or a control miRNA (miR CTRL) for 48 hrs and invasion measured overnight. <b>*</b> p<0.05 compared to miR control. (<b>F</b>) miR-194 expression in transiently transfected SKBr3 cells. SKBr3 cells were transiently transfected with a miR-194 precursor or a control miRNA (miR CTRL) for 48 hrs. Total RNA was extracted and subjected to QRT-PCR analysis for miR-194 expression. Hsa-miR-194 was purchased from ABI (Assay ID 000493). (<b>G</b>) Assay of cell migration in BT474 stable cells that express miR-194 or a control vector. The control clone #17 and the miR-194-expressing clone #22 were chosen to study migration. <b>*</b> p<0.05 compared to #17 control. (<b>H</b>) Cell invasion assay in BT474 stable cells that express miR-194 or its control vector. The control clone #17 and the miR-194-expressing clone #22 were chosen to study invasion. <b>*</b> p<0.05 compared to #17 control. (<b>I</b>) BT474 xenograft tumor growth in vivo. BT474 xenografts in nude mice were established with the control clone #17 and the miR-194-expressing clone #22 as described in Methods. Tumors were collected and weighed after 4 weeks. <b>*</b> p<0.05 compared to #17 control.</p