<i>In Vitro</i> Toxicity Assessment of Amphiphillic Polymer-Coated CdSe/ZnS Quantum Dots in Two Human Liver Cell Models

Semiconductor quantum dots (Qdots) are a promising new technology with benefits in the areas of medical diagnostics and therapeutics. Qdots generally consist of a semiconductor core, capping shell, and surface coating. The semiconductor core of Qdots is often composed of group II and VI metals (<i>e.g.</i>, Cd, Se, Te, Hg) that are known to have toxic properties. Various surface coatings have been shown to stabilize Qdots and thus shield cells from the toxic properties of their core elements. In this study, HepG2 cells and primary human liver (PHL) cells were chosen as <i>in vitro</i> tissue culture models of human liver to examine the possible adverse effects of tri-<i>n</i>-octylphosphine oxide, poly(maleic anhydride-<i>alt</i>-1-tetradecene) copolymer (TOPO-PMAT)-coated CdSe/ZnS Qdots (TOPO-PMAT Qdots). The TOPO-PMAT coating is desirable for increasing aqueous solubility and ease of conjugation to targeting moieties (<i>e.g.</i>, aptamers and peptides). HepG2 cells avidly incorporated these TOPO-PMAT Qdots into subcellular vesicles. However, PHL cells did not efficiently take up TOPO-PMAT Qdots, but nonparenchymal cells did (especially Kupffer cells). No acute toxicity or morphological changes were noted in either system at the exposure levels used (up to 40 nM). However, cellular stress markers and pro-inflammatory cytokines/chemokines were increased in the PHL cell cultures, suggesting that TOPO-PMAT Qdots are not likely to cause acute cytotoxicity in the liver but may elicit inflammation/hepatitis, demonstrating the importance of relevant preclinical safety models. Thus, further <i>in vivo</i> studies are warranted to ensure that TOPO-PMAT-coated Qdots used in biomedical applications do not induce inflammatory responses as a consequence of hepatic uptake

Amphiphilic polymer-coated CdSe/ZnS quantum dots induce pro-inflammatory cytokine expression in mouse lung epithelial cells and macrophages

<p>Quantum dots (Qdots) are semiconductor nanoparticles with size-tunable fluorescence capabilities with diverse applications. Qdots typically contain cadmium or other heavy metals, hence raising concerns of their potential toxicity, especially in occupational settings where inhalation of nanomaterials may increase the risk of lung disease. Accordingly, we assessed the effects of tri-<i>n</i>-octylphosphine oxide, poly(maleic anhydride-<i>alt</i>-1-tetradecene) (TOPO-PMAT) coated CdSe/ZnS Qdots on mouse lung epithelial cells and macrophages. Mouse tracheal epithelial cells (MTEC), grown as organotypic cultures, bone marrow-derived macrophages (BMDM), and primary alveolar macrophages (AM) were derived from C57BL/6J or A/J mice and treated with TOPO-PMAT CdSe/ZnS Qdots (10–160 nM) for up to 24 h. Cadmium analysis showed that Qdots remained in the apical compartment of MTEC cultures, whereas they were avidly internalized by AM and BMDM, which did not differ between strains. In MTEC, Qdots selectively induced expression (mRNA and protein) of neutrophil chemokines CXCL1 and CXCL2 but only low to no detectable levels of other factors assessed. In contrast, 4 h exposure to Qdots markedly increased expression of CXCL1, IL6, IL12, and other pro-inflammatory factors in BMDM. Higher inflammatory response was seen in C57BL/6J than in A/J BMDM. Similar expression responses were observed in AM, although overall levels were less robust than in BMDM. MTEC from A/J mice were more sensitive to Qdot pro-inflammatory effects while macrophages from C57BL/6J mice were more sensitive. These findings suggest that patterns of Qdot-induced pulmonary inflammation are likely to be cell-type specific and genetic background dependent.</p

The Glutathione Synthesis Gene <i>Gclm</i> Modulates Amphiphilic Polymer-Coated CdSe/ZnS Quantum Dot–Induced Lung Inflammation in Mice

<div><p>Quantum dots (QDs) are unique semi-conductor fluorescent nanoparticles with potential uses in a variety of biomedical applications. However, concerns exist regarding their potential toxicity, specifically their capacity to induce oxidative stress and inflammation. In this study we synthesized CdSe/ZnS core/shell QDs with a tri-n-octylphosphine oxide, poly(maleic anhydride-alt-1-tetradecene) (TOPO-PMAT) coating and assessed their effects on lung inflammation in mice. Previously published <i>in vitro</i> data demonstrated these TOPO-PMAT QDs cause oxidative stress resulting in increased expression of antioxidant proteins, including heme oxygenase, and the glutathione (GSH) synthesis enzyme glutamate cysteine ligase (GCL). We therefore investigated the effects of these QDs <i>in vivo</i> in mice deficient in GSH synthesis (<i>Gclm</i> +/− and <i>Gclm</i> −/− mice). When mice were exposed via nasal instillation to a TOPO-PMAT QD dose of 6 µg cadmium (Cd) equivalents/kg body weight, neutrophil counts in bronchoalveolar lavage fluid (BALF) increased in both <i>Gclm</i> wild-type (+/+) and <i>Gclm</i> heterozygous (+/−) mice, whereas <i>Gclm</i> null (−/−) mice exhibited no such increase. Levels of the pro-inflammatory cytokines KC and TNFα increased in BALF from <i>Gclm</i> +/+ and +/− mice, but not from <i>Gclm</i> −/− mice. Analysis of lung Cd levels suggested that QDs were cleared more readily from the lungs of <i>Gclm</i> −/− mice. There was no change in matrix metalloproteinase (MMP) activity in any of the mice. However, there was a decrease in whole lung myeloperoxidase (MPO) content in <i>Gclm</i> −/− mice, regardless of treatment, relative to untreated <i>Gclm</i> +/+ mice. We conclude that in mice TOPO-PMAT QDs have <i>in vivo</i> pro-inflammatory properties, and the inflammatory response is dependent on GSH synthesis status. Because there is a common polymorphism in humans that influences GCLM expression, these findings imply that humans with reduced GSH synthesis capabilities may be more susceptible to the pro-inflammatory effects of QDs.</p></div

mRNA expression of inflammatory cytokine genes as measured by qRT-PCR.

<p>The effects of saline or TOPO-PMAT QD administration on A) <i>Il1β</i>, B) <i>Mcp1</i>, C) <i>Tnfα</i>, D) <i>Gmcsf</i> and E) <i>KC</i> mRNA expression are shown. Data represent the mean ± S.E.M. of the indicated number of replicates in each bar.</p

mRNA expression of stress-response genes as measured by qRT-PCR.

<p>The effects of saline and TOPO-PMAT QD administration on A) <i>Gclc</i>, B) <i>Gclm</i>, C) <i>Hmox1</i>, D) <i>Mt1</i> and E) <i>Mt2</i> mRNA expression are shown. Data represent the mean ± S.E.M., * = p<0.05 relative to saline treated <i>Gclm</i> +/− mice. The number of replicates is indicated in each bar.</p

Toxicokinetic modeling of TOPO-PMAT QD disposition.

<p>Lung cadmium content was determined by inductively coupled plasma-mass spectrometry (ICP-MS) analysis, and TOPO-PMAT QD disposition was modeled using PKSolver as described in Methods. A one-compartment model resulted in the best fit of simulated QD disposition curves relative to measured Cd content.</p

Lung matrix metalloproteinase activity and myeloperoxidase content.

<p>The effect of TOPO-PMAT QD administration on A) <b>matrix metalloproteinase (</b>MMP) activity and B) <b>myeloperoxidase</b> (MPO) content. Data represent the mean ± S.E.M. of 4 replicates ** = p<0.01 relative to saline treated mice.</p

Schematic representation of a TOPO-PMAT quantum dot.

<p>The central sphere represents the CdSe/ZnS core/cap structure, surrounded next by tri-n-octylphosphine oxide (TOPO), and then the poly(maleic anhydride-alt-1-tetradecene (PMAT) polymer coating. (Modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064165#pone.0064165-Smith1" target="_blank">[42]</a>).</p

% Neutrophil influx into the bronchoalveolar lavage fluid.

<p>Data represent the mean ± S.E.M. for % neutrophils (cells exhibiting low F4/80 and high Gr1 staining) present in the bronchoalveolar lavage fluid as measured by flow cytometry 8 hours following saline or TOPO-PMAT QD administration. * = p<0.05 relative to saline treated mice. The number of replicates is indicated in each bar.</p

Inflammatory cytokine protein expression in lung tissue.

<p>The effect of saline or TOPO-PMAT QD administration on A) KC and B) TNFα protein levels as assessed in a cytokine array panel. Data represent the mean ± S.E.M. of the indicated number of replicates in each bar. * = p<0.05 relative to saline treated mice.</p