Background: Nanomaterials with particle sizes <100 nm have been already applied in various applications such as cosmetics, medicines, and foods. Therefore, ensuring the safety of nanomaterials is becoming increasingly important. Here we examined the localization and biological responses of intranasally administered amorphous nanosilica particles in mice, focusing on the coagulation system. Methods: We used nanosilica particles with diameters of 30, 70, or 100 nm (nSP30, nSP70, or nSP100 respectively), and conventional microscale silica particles with diameters of 300 or 1000 nm (mSP300 or mSP1000, respectively). BALB/c mice were intranasally exposed to nSP30, nSP70, nSP100, mSP300, or mSP1000 at concentrations of 500 μg/mouse for 7 days. After 24 hours of last administration, we performed the in vivo transmission electron microscopy analysis, hematological examination and coagulation tests. Results: In vivo transmission electron microscopy analysis showed that nanosilica particles with a diameter <100 nm were absorbed through the nasal cavity and were distributed into liver and brain. Hematological examination and coagulation tests showed that platelet counts decreased and that the activated partial thromboplastin time was prolonged in nSP30 or nSP70-treated groups of mice, indicating that nanosilica particles might have activated a coagulation cascade. In addition, in in vitro activation tests of human plasma, nanosilica particles had greater potential than did conventional microscale silica particles to activate coagulation factor XII. In nanosilica-particle-treated groups, the levels of soluble CD40 ligand, and von Willebrand factor which are involved in stimulating platelets tended to slightly increase with decreasing particle size. Conclusions: These results suggest that intranasally administered nanosilica particles with diameters of 30 and 70 nm could induce abnormal activation of the coagulation system through the activation of an intrinsic coagulation cascade. This study provides information to advance the development of safe and effective nanosilica particles.

With the increase in use of nanomaterials, there is growing concern regarding their potential health risks. However, few studies have assessed the role of the different physical characteristics of nanomaterials in allergic responses. Here, we examined whether intranasally administered silica particles of various sizes have the capacity to promote allergic immune responses in mice. We used nanosilica particles with diameters of 30 or 70 nm (nSP30 or nSP70, respectively), and conventional micro-sized silica particles with diameters of 300 or 1000 nm (nSP300 or mSP1000, respectively). Mice were intranasally exposed to ovalbumin (OVA) plus each silica particle, and the levels of OVA-specific antibodies (Abs) in the plasma were determined. Intranasal exposure to OVA plus smaller nanosilica particles tended to induce a higher level of OVA-specific immunoglobulin (Ig) E, IgG and IgG1 Abs than did exposure to OVA plus larger silica particles. Splenocytes from mice exposed to OVA plus nSP30 secreted higher levels of Th2-type cytokines than mice exposed to OVA alone. Taken together, these results indicate that nanosilica particles can induce allergen-specific Th2-type allergic immune responses in vivo. This study provides the foundations for the establishment of safe and effective forms of nanosilica particles.

Although amorphous silica nanoparticles are widely used in the production of food products (e.g., as anticaking agents), there is little information available about their absorption and biological effects after oral exposure. Here, we examined the in vitro intestinal absorption and in vivo biological effects in mice of orally administered amorphous silica particles with diameters of 70, 300, and 1,000 nm (nSP70, mSP300, and mSP1000, respectively) and of nSP70 that had been surface-modified with carboxyl or amine groups (nSP70-C and nSP70-N, respectively). Analysis of intestinal absorption by means of the everted gut sac method combined with an inductively coupled plasma optical emission spectrometer showed that the intestinal absorption of nSP70-C was significantly greater than that of nSP70. The absorption of nSP70-N tended to be greater than that of nSP70; however, the results were not statistically significant. Our results indicate that silica nanoparticles can be absorbed through the intestine and that particle diameter and surface properties are major determinants of the degree of absorption. We also examined the biological effects of the silica particles after 28-day oral exposure in mice. Hematological, histopathological, and biochemical analyses showed no significant differences between control mice and mice treated with the silica particles, suggesting that the silica nanoparticles evaluated in this study are safe for use in food production.

Nanomaterials are used for various biomedical applications because they are often more effective than conventional materials. Recently, however, it has become clear that the protein corona that forms on the surface of nanomaterials when they make contact with biological fluids, such as blood, influences the pharmacokinetics and biological responses induced by the nanomaterials. Therefore, when evaluating nanomaterial safety and efficacy, it is important to analyze the interaction between nanomaterials and proteins in biological fluids and to evaluate the effects of the protein corona. Here, we evaluated the interaction of silica nanoparticles, a commonly used nanomaterial, with the human blood proteins albumin, transferrin, fibrinogen, and IgG. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the amount of albumin, transferrin, and IgG binding to the silica particles increased as the particle size decreased under conditions where the silica particle mass remained the same. However, under conditions in which the specific surface area remained constant, there were no differences in the binding of human plasma proteins to the silica particles tested, suggesting that the binding of silica particles with human plasma proteins is dependent on the specific surface area of the silica particles. Furthermore, the amount of albumin, transferrin, and IgG binding to silica nanoparticles with a diameter of 70 nm (nSP70) and a functional amino group was lower than that with unmodified nSP70, although there was no difference in the binding between nSP70 with the surface modification of a carboxyl functional group and nSP70. These results suggest that the characteristics of nanomaterials are important for binding with human blood proteins; this information may contribute to the development of safe and effective nanomaterials.

The prevalent use of engineered nanoparticles (ENPs) has increased our exposure to these particles. The current available analytical techniques fail to simultaneously quantify and analyze the physical properties of ENPs in biological tissues. Therefore, new methods are required to evaluate the exposure conditions to ENPs. Single particle inductively coupled plasma-mass spectrometry (sp-ICP-MS) is an attractive approach that can perform quantitative and qualitative analyses of ENPs. However, the application of this approach for biological samples is limited because of the lack of pretreatment methods for effectively recovering ENPs from biological tissues. In this study, we evaluated various pretreatment methods and identified the optimal pretreatment conditions for sp-ICP-MS analyses of ENPs in biological tissues using silver nanoparticles (nAg) as a model. We screened five reagents as pretreatment solvents (sodium hydroxide, tetramethylammonium hydroxide, nitric acid, hydrochloric acid, and proteinase K). Our results showed that treatment with sodium hydroxide was optimal for detecting nAg in the mouse liver. Moreover, this pretreatment method can be applied to other organs, such as the heart, lung, spleen, and kidney. Finally, we evaluated the applicability of this method by analyzing the quantity and physical properties of silver in the mouse blood and liver, after intravenous administration of nAg or silver ion. Our sp-ICP-MS method revealed that nAg administered into the blood was partially ionized and tended to be distributed in the particle form (approximately 80%) in the liver and in ionic form (approximately 95%) in the blood. In conclusion, we optimized pretreatment strategies for sp-ICP-MS evaluation of ENPs in biological tissues and demonstrated its applicability by evaluating the changes in the physical properties of nAg in the liver and blood. We also showed that partial changes from the particle form to the ionic form of nAg influences their kinetics and distribution when administered to mice.