Aerosol-to-liquid collection: A method for making aqueous suspension of hydrophobic nanomaterial without adding dispersant

<p>This article introduces an aerosol-based technique to make aqueous suspension of hydrophobic nanomaterial without adding dispersant. The method is intended for making a test-sample for evaluating the toxicities of nanomaterial by intra-tracheal administration. The method can wet the surface of hydrophobic nanomaterial within a few seconds. After the wetting process five to ten minutes of sonication assisted with manual stirring can fully disperse the hydrophobic nanomaterials in water. Two types of TiO₂ nanomaterial were used in this study; Tayca JMT-150IB whose surfaces are coated with negatively charged hydrophobic functional group, and P25 whose surfaces are naturally hydrophilic. Nanomaterials are aerosolized by a dry-method and become micrometer-sized agglomerates. Then supersaturated water vapor is condensed onto these airborne agglomerates by using a growth tube collector. The collected suspension (CS) of hydrophobic nanomaterial (JMT-150IB) is prepared in two steps; airborne agglomerates are collected onto a flat surface then transferred to liquid-water and subsequently sonicated for complete dispersion. This method works equally well for making the CS of hydrophilic nanomaterial. Size distribution measurements of the CS show that airborne agglomerates of TiO₂ dissociate into smaller units of agglomerates once they are captured into water, and the sizes of the agglomerates are in the nanometer to sub-micrometer range. Light scattering technique is used to show that a short sonication process can reproduce the particle number concentration of the CS after long storage.</p> <p>Copyright © 2017 American Association for Aerosol Research</p

Sequence of the primers for qRT-PCR.

<p>Sequence of the primers for qRT-PCR.</p

Chemical structure of α-adrenoceptor antagonists used.

<p>Chemical structure of α-adrenoceptor antagonists used.</p

Sequence of the primers for cloning of Cited4.

<p>Sequence of the primers for cloning of Cited4.</p

The <i>Cited4</i> gene is expressed specifically in cardiac progenitor cell populations during <i>in vitro</i> cardiogenesis.

<p>(A) Time-dependent expression of GFP under the control of a Rex1-promoter was evaluated at day 0 of undifferentiation embryonic stem cells and day 3.5 in the EBs formed with the Rex1-ht7 cell line. Scale bar = 100 μm. (B) Time-dependent expression of GFP under the control of a Bra-promoter was evaluated at day 3.5, day 4.5, day 5.5, and day 7.5 in the EBs formed with the Bra-ht7 cell line. Scale bar = 100 μm. (C) To obtain the pluripotent embryonic stem cell population, the Rex1 and E-cadherin double positive cell population was isolated from the Rex1-ht7 cell line at day 0 before forming the EBs. (D) To obtain the primitive mesodermal cell population and the early lateral mesodermal cell population, the Bra-positive and Flk1-negative cell population and the Bra-negative and Flk1-positive cell population were isolated from the Bra-ht7 cell line at day 4.5 in the EBs, respectively. (E) To obtain the cardiac progenitor cell population, the Nkx2.5-positive and Flk1-negative cell population was isolated from the hcgp7 cell line at day 7.5 in the EBs. (F) The <i>Cited4</i> gene expressions in the isolated lineage marker-positive cell populations were evaluated by qRT-PCR and normalized to <i>β-actin</i> mRNA levels (N = 3). Values are presented as the mean ± SEM. N: number of experiments. *<i>P</i> < 0.05 vs. Rex1-positive embrynic stem cells.</p

List of antibodies for flow cytometry.

<p>List of antibodies for flow cytometry.</p

Time course of accumulation of Hoechst33342.

<p>Cells were incubated with 3 µM Hoechst33342 in the absence (○) or presence of α-adrenoceptor antagonists (ergot alkaloids: ▴, 1 nM; ▪, 10 nM; ♦, 100 nM, other α-adrenoceptor antagonists: ▴, 0.1 µM; ▪, 1 µM; ♦, 10 µM) for the desired times at 37°C. Each point represents the mean ± S.E. (n = 3), and error bars are included in the symbols. * and ** p<0.05 and 0.01 significantly different from the control at the corresponding time points, respectively.</p

Cell cycle profile of HeLa/SN100 cells treated with mitoxantrone in the presence of α-adrenoceptor antagonists.

<p>HeLa/SN100 cells were incubated with mitoxantrone for 8 hours in the absence (Control) or presence of α-adrenoceptor antagonists before being stained with propidium iodide. DNA content was analyzed by flow-cytometry and the percentage of cells in each phase of the cell cycle was obtained by using the Modfit program.</p

Time course of the efflux of Hoechst33342.

<p>Cells were incubated with 3 µM Hoechst33342 in the presence of 10 µM of α-adrenoceptor antagonists (100 nM for ergot alkaloids) for 60 min at 37°C, washed twice with ice-cold HBSS and incubated with warmed HBSS in the presence of 10 µM of α-adrenoceptor antagonists (100 nM for ergot alkaloids) for the desired times. ○, HeLa; •, HeLa/SN100; Δ, HeLa/SN100+10 µM α-adrenoceptor antagonists (100 nM for ergot alkaloids). Each point represents the mean ± S.E. (n = 3), and error bars are included in the symbols. * and ** p<0.05 and 0.01 significantly different from the HeLa/SN100 cells in the absence of α-adrenoceptor antagonists at the corresponding time points, respectively.</p

The <i>Cited4</i> gene is expressed transiently during the early cardiac stage of <i>in vitro</i> cardiogenesis.

<p>(A-D) mRNA expression levels of lineage marker genes during <i>in vitro</i> cardiogenesis were measured by qRT-PCR and normalized to <i>β-actin</i> mRNA levels. <i>Rex1</i>, <i>Bra</i>, <i>Flk1</i>, and <i>Nkx2</i>.<i>5</i> genes were lineage markers for undifferentiated pluripotent stem cells, early primitive mesodermal cells, lateral mesodermal cells, and cardiac progenitor cells, respectively (N = 4). (E) The proportion of beating EBs, a classical <i>in vitro</i> cardiogenesis marker, was evaluated during <i>in vitro</i> cardiogenesis (N = 3, n = 240). (F) mRNA expression levels of the <i>Cited4</i> gene during <i>in vitro</i> cardiogenesis were measured by qRT-PCR and normalized to <i>β-actin</i> mRNA levels (N = 3). N: number of experiments; n: total number of EBs counted. *<i>P</i> < 0.05 vs. <i>Rex1</i> expression at day 4.5, <i>Bra</i> expression at day 3.5, <i>Flk1</i> expression at day 0, <i>Nkx2</i>.<i>5</i> expression at day 0, <i>Cited4</i> expression at day 0, and proportion of beaing EBs at day 5.0.</p