High inflammogenic potential of rare earth oxide nanoparticles: the New Hazardous Entity

<p>Due to the exponential increase in the development and utilization of rare earth oxide nanoparticles (REO NPs) in various fields, the possibility of exposure in humans by inhalation has increased. However, there are little information about hazards of REO NPs and its mechanisms of toxicity. In this study, we evaluated the acute pulmonary inflammation using 10 REO NPs (Dy₂O₃, Er₂O₃, Eu₂O₃, Gd₂O₃ La₂O₃, Nd₂O₃, Pr₆O₁₁, Sm₂O₃, Tb₄O₇, and Y₂O₃) and four well-known toxic particles (CuO, NiO, ZnO, and DQ12). Minimum three doses per NP were instilled into the lungs of female Wistar rats at surface area dose metric and lung inflammation was evaluated at 24 h post-instillation by bronchoalveolar lavage fluid (BALF) analysis and histopathological observation. All types of REO NPs showed common pathological changes including mild to moderate infiltration of neutrophils and activated macrophages in the alveoli, peribronchial, and perivascular region. The inflammogenic potential evaluated by the number of granulocytes divided by the treated surface area dose showed all types of REO NPs has much higher inflammogenic potential than DQ12, ZnO, and NiO NPs. The correlation plot between the number of granulocytes and the potential for reactive oxygen species (ROS) generation showed a good correlation with exception of Pr₆O₁₁. The higher inflammogenic potential of REO NPs than that of well-known highly toxic particles imply that REO NPs need special attention for inhalation exposure and more studies are needed. In addition, the potential of ROS generation is one of the key factors producing lung inflammation by REO NPs.</p

Surface charge determines the lung inflammogenicity: A study with polystyrene nanoparticles

<p>Surface functionalization is a routine process to improve the behavior of nanoparticles (NPs), but the induced surface properties, such as surface charge, can produce differential toxicity profiles. Here, we synthesized a library of covalently functionalized fluorescent polymeric NPs (<i>F-</i>PLNPs) to evaluate the role of surface charge on the acute inflammation and the localization in the lung. Guanidinium-, acetylated-, zwitterionic-, hydroxylated-, PEGylated-, carboxylated- and sulfated-<i>F-</i>PLNPs were synthesized from aminated-<i>F</i>-PLNP. The primary particle sizes were identical, but the hydrodynamic sizes ranged from 210 to 345 nm. Following surface functionalization, the <i>F</i>-PLNPs showed diverse zeta potentials from −41.2 to 31.0 mV, and each <i>F</i>-PLNP showed a single, narrow peak. Pharyngeal aspiration with these eight types of <i>F</i>-PLNPs into rats produced diverse acute lung inflammation, with zeta potentials of the <i>F</i>-PLNPs showing excellent correlation with acute pulmonary inflammation parameters including the percentage of polymorphonuclear leukocytes (<i>R</i>² = 0.90, <i>p</i> < 0.0001) and the levels of interleukin-1β (<i>R</i>² = 0.83, <i>p</i> < 0.0001) and of cytokine-induced neutrophil chemoattractant-3 (<i>R</i>² = 0.86, <i>p</i> < 0.0001). These results imply that surface charge is a key factor influencing lung inflammation by functionalized polymeric NPs, which further confirms and extends the surface charge paradigm that we reported for pristine metal oxide NPs. This demonstrates that the surface charge paradigm is a valuable tool to predict the toxicity of NPs.</p

Surface functionalization-specific binding of coagulation factors by zinc oxide nanoparticles delays coagulation time and reduces thrombin generation potential <i>in vitro</i>

<div><p>Zinc oxide nanoparticles (ZnO NPs) have many biomedical applications such as chemotherapy agents, vaccine adjuvants, and biosensors but its hemocompatibility is still poorly understood, especially in the event of direct contact of NPs with blood components. Here, we investigated the impact of size and surface functional groups on the platelet homeostasis. ZnO NPs were synthesized in two different sizes (20 and 100 nm) and with three different functional surface groups (pristine, citrate, and L-serine). ZnO NPs were incubated with plasma collected from healthy rats to evaluate the coagulation time, kinetics of thrombin generation, and profile of levels of coagulation factors in the supernatant and coronated onto the ZnO NPs. Measurements of plasma coagulation time showed that all types of ZnO NPs prolonged both active partial thromboplastin time and prothrombin time in a dose-dependent manner but there was no size- or surface functionalization-specific pattern. The kinetics data of thrombin generation showed that ZnO NPs reduced the thrombin generation potential with functionalization-specificity in the order of pristine > citrate > L-serine but there was no size-specificity. The profile of levels of coagulation factors in the supernatant and coronated onto the ZnO NPs after incubation of platelet-poor plasma with ZnO NPs showed that ZnO NPs reduced the levels of coagulation factors in the supernatant with functionalization-specificity. Interestingly, the pattern of coagulation factors in the supernatant was consistent with the levels of coagulation factors adsorbed onto the NPs, which might imply that ZnO NPs simply adsorb coagulation factors rather than stimulating these factors. The reduced levels of coagulation factors in the supernatant were consistent with the delayed coagulation time and reduced potential for thrombin generation, which imply that the adsorbed coagulation factors are not functional.</p></div

Measurement of active partial thromboplastin time and prothrombin time after incubation of platelet-poor plasma and ZnO NPs.

<p>aPTT was measured for (A) 20 nm- and (B) 100 nm-sized ZnO NPs. PT was measured for (C) 20 nm- and (D) 100 nm-sized ZnO NPs. ZnO NPs were conjugated with none (pristine), citrate, and L-serine to provide three distinctive charged surfaces. Each sample was analyzed in duplicate and repeated three times, by using three separate plasma sets. Results are means ± SEM, *<i>p</i> < 0.05 versus control.</p

Thrombin generation assay of ZnO NPs in platelet poor plasma.

<p>Thrombin generation assay was performed with 20 nm-sized ZnO NPs at (A) 0.1, (B) 0.25, and (C) 0.5 mg/mL. Thrombin generation assay was also performed with 100 nm-sized ZnO NPs at (C) 0.1, (D) 0.25, and (E) 0.5 mg/mL. <i>n</i> = 3.</p

Western blot analysis of ZnO NP-bound coagulation factors on the surface of NPs.

<p>ZnO NPs were incubated with PPP and NP pellets were collected and dissolved in loading buffer, then analyzed for coagulation factors using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting. Each factor was separately subjected to densitometric analysis.</p

Summary of expression profile of coagulation factors observed by ELISA and western blot analysis.

<p>Summary of expression profile of coagulation factors observed by ELISA and western blot analysis.</p

Physicochemical properties of ZnO NP.

<p>Physicochemical properties of ZnO NP.</p

The levels of coagulation factors in the supernatant of platelet poor plasma after incubation with ZnO NPs.

<p><b>ZnO NPs at 0.5 mg/mL were incubated with PPP and the levels of coagulation factors were measured using ELISA kits in the NP-free supernatant.</b> (A), factor II; (B), factor III; (C), factor V; (D), factor VII; (E), factor VIII; (F), factor IX; (G), factor X; (H), factor XI; (I), factor XII. *<i>p</i> < 0.05 and <i>n</i> = 4.</p

Scanning electron microscopy (SEM) images of ZnO NPs.

<p>(A) Pristine 20 nm, (B) Citrate 20 nm, (C) L-serine 20 nm, (D) Pristine 100 nm, (E) Citrate 100 nm, and (F) L-serine 100 nm.</p