Centrifugal Field-Flow Fractionation Enables Detection of Slight Aggregation of Nanoparticles That Impacts Their Biomedical Applications

Nanoparticles (NPs) are anticipated to be used for various biomedical applications in which their aggregation has been an important issue. However, concerns regarding slightly aggregated but apparently monodispersed NPs have been difficult to address because of a lack of appropriate evaluation methods. Here, we report centrifugal field-flow fractionation (CF3) as a powerful method for analyzing the slight aggregation of NPs, using antibody-modified gold NPs (Ab-AuNPs) prepared by a conventional protocol with centrifugal purification as a model. While common evaluation methods such as dynamic light scattering cannot detect significant signs of aggregation, CF3 successfully detects distinct peaks of slightly aggregated NPs, including dimers and trimers. Their impact on biological interactions was also demonstrated by a cellular uptake study: slightly aggregated Ab-AuNPs exhibited 1.8 times higher cellular uptake than monodispersed Ab-AuNPs. These results suggest the importance of aggregate evaluation via CF3 as well as the need for careful attention to the bioconjugation procedures for NPs

Early Postnatal Exposure to a Low Dose of Decabromodiphenyl Ether Affects Expression of Androgen and Thyroid Hormone Receptor-Alpha and Its Splicing Variants in Mouse Sertoli Cells

<div><p>Decabromodiphenyl ether (decaBDE) adversely affects reproduction and development. Our previous study showed that postnatal exposure to a low dose of decaBDE (0.025 mg/kg body weight/day) by subcutaneous injection on postnatal days (PNDs) 1 through 5 leads to reductions in testicular size and number of Sertoli cells and sperm, while higher dose of decaBDE (2.5 mg/kg body weight/day) had no significant differences about these. In the present study, we examined the molecular mechanism of these effects on mouse testes following postnatal exposure to a low decaBDE dose. We hypothesized that postnatal exposure to decaBDE may alter levels of serum thyroid hormones (THs) and testosterone, or the level of TH receptor alpha (<i>Thra</i>) transcripts and its splicing variants and androgen receptor (<i>Ar</i>) in Sertoli cells, adversely affecting spermatogenesis. To test this hypothesis, we examined serum TH and testosterone levels and the levels of transcripts of the <i>Ar, Thra</i> and its splicing variants, and <i>Thra</i> splicing factors (<i>Hnrnpa1</i>, <i>Srsf1</i>, and <i>Hnrnph1</i>) with qPCR in isolated mouse Sertoli cells exposed postnatally to decaBDE (0.025, 0.25, and 2.5 mg/kg). Levels of serum testosterone and transcripts encoding <i>Ar</i>, <i>Thra</i>, and its variant, <i>Thra1,</i> declined significantly in Sertoli cells of mice exposed to 0.025 mg decaBDE/kg. No significant differences in serum TH level or <i>Thra2, Hnrnph1</i>, or <i>Srsf1</i> transcript levels were observed between control and decaBDE-exposed mice. However, the <i>Thra1</i>:<i>Thra2</i> and <i>Hnrnpa1</i>:<i>Srsf1</i> ratios were altered in Sertoli cells of mice exposed to 0.025 mg decaBDE/kg but not in cells exposed to 0.25 or 2.5 mg decaBDE/kg. These results indicate that postnatal exposure to a low dose of decaBDE on PNDs 1 through 5 lowers the testosterone level and the levels of <i>Ar</i> and <i>Thra</i> transcripts in Sertoli cells, accompanied by an imbalance in the ratios of <i>Thra</i> splicing variants, resulting in smaller testicular size and impaired spermatogenesis.</p></div

Possible mechanism following early postnatal exposure to a low dose of decaBDE (0.025 mg/kg) in mouse testis.

<p>Early postnatal exposure to decaBDE from PND 1 to 5 causes to reduce serum testosterone level and <i>Ar</i> transcript levels in Sertoli cells, resulting in smaller testes size, reduced sperm count and a lower number of Sertoli cells, as well as decreased transcript levels of <i>Thra</i> and its splicing variant, <i>Thra1,</i> and an imbalance in the expression ratios of <i>Thra1</i>:<i>Thra2</i> and <i>Hnrnpa1</i>:<i>Srsf1</i> (gray box). It remains unknown if these latter changes are related to smaller testes size, reduced sperm count and lower number of Sertoli cells (dotted line).</p

Levels of <i>Thra</i> and <i>Ar</i> transcripts in isolated Sertoli cells.

<p>Levels of transcripts of <i>Thra</i> (A), <i>Thra1</i> (B), <i>Thra2</i> (C), and <i>Ar</i> (D) were measured using qPCR in control and decaBDE-exposed isolated Sertoli cells cultured for 1 week. Transcript expression was normalized to the level of <i>Actb</i> transcript expression and is shown as the ratio relative to <i>Actb</i> compared with the control (set as a value of 1.0). Each value is the mean ± SD of 6-9 samples per group. *<i>P</i><0.05 compared with the control.</p

Serum TH levels in control and decaBDE-exposed mice.

<p>Total T₃ (A), total T₄ (B), FT₃ (C) and FT₄ (D) levels in the serum of control and decaBDE-exposed mice were measured by ELISA. Each value is the mean ± SD of 6 samples per group.</p

Serum testosterone levels in control and decaBDE-exposed mice.

<p>Testosterone levels in the serum of control and decaBDE-exposed mice were measured by ELISA. Each value is the mean ± SD of 6 samples per group. *<i>P</i><0.05 compared with the control.</p

Schematic illustrations of phage display screening method.

The hepatitis C virus (HCV) E2 protein derived from either strain TH (genotype 1b) or strain JFH-1 (genotype 2a) was allowed to bind to the surface of magnetic beads, and the single chain Fv (scFv) phage library then was added to the coated beads. Specifically bound phages were amplified by infection of E. coli. These steps were repeated for a total of 4 times to enrich for phages with specific binding to the HCV E2 protein. To confirm the specific binding of scFv phages to HCV E2 protein-bound magnetic beads, the gene encoding the corresponding IgG was constructed from phagemid scFv-encoding sequences by PCR cloning. Specific binding of these antibodies to HCV E2 protein was assessed by enzyme immune assay (EIA) recognition and neutralization of HCV pseudoparticle (HCVpp) infection. (TIF)</p

Results of phage display screen.

Results of phage display screen.</p

List of antibodies.

(DOCX)</p

Neutralizing activities of phage library-derived IgGs against HCVpp infection.

The neutralization effects of the e2d066, e2d073, and e2d081 antibodies were assessed by infection assays using HCV pseudoparticles (HCVpp) harboring the E1 and E2 glycoproteins of (A) TH (genotype 1b) and (B) J6CF (genotype 2a). HCVpp, which include a luciferase-encoding construct, were mixed with immunoglobulin G (IgG) or phosphate buffered saline (PBS) for 30 minutes at room temperature; the mixtures then were used to inoculate naïve Huh-7.5.1 cells. Monoclonal anti-CD81 antibody (JS-81, BD Pharmingen) was used as positive control in this assay. At 72 hours after infection, the infected cells were harvested and lysed with Cell Culture Lysis Reagent (Promega). Luciferase activity was quantified using the Luciferase Assay System (Promega). Neutralizing activity was calculated and is presented as the % neutralization by comparison with the luciferase activities of the well inoculated with the HCVpp-PBS mixture. Assay were performed in triplicate and infection rate are expressed as mean ± SEM. Statistical significance of difference was analyzed using one-way ANOVA with a Williams’ test (*P < 0.025, **P < 0.005, ***P < 0.0005 vs PBS).</p