Merget et al. suggested that the inhalation of amorphous silica particles in the workplace could induce silicosis . In addition, nanosilica particles have been explored for medical applications, such as cancer therapeutics or drug-delivery agents via intranasal administration [23, 24]. However, there is not enough information about the biological effects of nanosilica particles after intranasal exposure for them to be used safely. In this study, we focused on intranasal exposure of nanosilica particles and determined the localization and biological effects of nanosilica particles after intranasal administration.
Warheit et al. and Lee et al. reported that the no-observable-effect level (NOEL) of colloidal silica particles is 489 μg/lung (equivalent to an inhalation exposure of 10 mg/m3) [25, 26]. Accordingly, we designed our experiments such that mice were intranasally exposed to various sizes of silica particles at 500 μg/mouse for 7 days, a level close to the NOEL for inhalation exposure. The dose in our study is important from the viewpoint of establishing an upper threshold for the amount of NMs that can safely be administered intranasally. Although we still need to accumulate much more information about the biological effects of nanosilica particles using intranasal administration, at realistic exposure levels, we expect that our present study will contribute to the safety assessment of NMs.
We found that nSP30, nSP70, and nSP100 were located not only in the nasal cavity and lung but also in the liver (Figure 1). In our previous study, we showed that nSP70, mSP300, and mSP1000 localized in the liver after entering the bloodstream . When we hypothesize how the nanosilica particles (smaller than 100 nm) enter the liver after intranasal administration, it is important to discuss how nanosilica particles enter the bloodstream through the nasal cavity or lung. We hypothesized that the nanosilica particles were absorbed through transcytosis, or uptake by microfold cells (M cells) in bronchus-associated lymphoid tissues and nasal-associated lymphoid tissues. Other reports have suggested that NMs open the tight junction, which plays an important role in maintaining the epithelial barrier [27, 28]. Thus, to investigate the pathway by which nanosilica particles enter the body, we need to evaluate the effects of nanosilica particles on M cells or epithelial cell barriers in vitro. In this study, we examined only the nasal cavity, lung, and liver, so we cannot comment as to whether nanosilica particles are localized in other tissues. However, other groups have reported that titanium dioxide nanoparticles with a diameter of 80 nm were localized in the brain after intranasal administration [29, 30]. Furthermore, Liu et al. showed that copper nanoparticles with a diameter of 23.5 nm enter the olfactory bulb in the brain . Therefore, in our study, it is possible that nanosilica particles with diameters of 30 or 70 nm may have been localized in the brain. In this analysis, the particles detected in tissues after intranasal administration of mSP300 and mSP1000 were smaller than the average diameter of the respective administered particles (Figure 1a,b,e). We consider the possibility of degradation of mSP300 and mSP1000 in the body, because some previous in vitro studies have suggested that silica particles could be degraded in humans and animals after absorption [32–34]. For example, Kim et al. showed that approximately 50% to 80% of a sample of microsilica particles was dissolved within 36 h in a solution of phosphate-buffered saline with 10% bovine serum, which is a simulated body fluid . In addition, a recent study suggested the possibility of silica particle degradation at a cellular level. Zhai et al. showed that hollow mesoporous nanosilica particles degraded when injected into human umbilical vein endothelial cells . On the basis of these reports’ findings, silica particles localized in biological bodies might be degraded within 7 days by means of interaction with biological fluid or by uptake into epithelial cells in the nasal cavity or lungs. On the other hand, because transmission electron microscopy analysis is only a qualitative method, we need to quantitatively analyze the silica particles after intranasal exposure to obtain more detailed information about their biodistribution. Inductively coupled plasma–optical emission spectrometry (ICP-OES) is reported to be a suitable means for quantitatively measuring silica. Using ICP-OES, we initially attempted to quantify the absorption of nSP30 and nSP70 in the liver after intranasal exposure for 7 days. However, we did not detect the particles in biological tissue using ICP-OES (data not shown; the detection limit of our protocol was 50 μg/g). In our study, nanosilica particles were not localized in the liver at levels sufficient for measurement by ICP-OES. To quantitatively analyze the nanosilica particles and clarify their absorption, distribution, metabolism, and excretion mechanisms, a method with greater sensitivity must be developed.
Previously, we found that nanosilica particles could accumulate in the liver and induce severe liver damage after intravenous administration [11, 15]. The level of nanosilica particles accumulated in the liver after intranasal administration would be lower than that observed after intravenous administration, and thus a nanosilica-particle-mediated increase of ALT levels or abnormal findings in pathological examination would have been reduced in the present study. We must measure the level of nanosilica particles in the liver quantitatively to confirm this speculation; overall, our present findings suggest that we need to more precisely evaluate the biological effects of intranasally administered NMs on all tissues in the body, including the liver and brain.
Intranasally administered nanosilica particles might have induced abnormal activation of the intrinsic coagulation cascade (Figures 4 and 5). To explain the decrease of platelets observed in the nanosilica-particle-treated groups, the nanosilica particles might directly activate the platelets and promote the coagulation cascade, resulting in consumption of platelets and consequently prolonged bleeding times. Other groups have shown that some NMs, such as single-walled carbon nanotubes and rutile titanium dioxide nanorods, could activate platelets and induce abnormal activation of the coagulation system [35–38]. Therefore, in our study the platelets might have been activated by the nanosilica particles and then subsequently consumed as they formed blood clots, thus decreasing the number of platelets and consequently prolonging bleeding time. Furthermore, the platelet counts in the nSP70-treated group at concentrations <250 μg/mouse and in the nSP30-treated group at concentrations <62.5 μg/mouse were equal to the count of the control group. Thus, this finding could provide useful information for setting the no-observable-adverse-effect level for intranasally administered nanosilica particles. Our present results indicate that the abnormal activation of a coagulation cascade by nanosilica particles after intranasal administration was promoted by activation of an intrinsic cascade pathway (Figure 5). However, our previous study showed that intravenously administered nSP70 could induce the release of TF, which is a known marker of activation of an extrinsic cascade pathway . We speculate that the level of intranasally administered nanosilica particles in the bloodstream was lower than that of intravenously administered nanosilica particles, and thus a drastic release of TF was not detected in this study.
Contact activation of coagulation factor XII is one of the major factors of blood coagulation [39, 40], and, as mentioned earlier, coagulation factor XII is activated when it comes into contact with hydrophilic activating particles (such as fully water-wettable glass) . Since the number of silica particles per unit weight increases as the particle size decreases (the particle numbers of the silica particles were 3.5 × 1013, 2.8 × 1012, 9.5 × 1011, 3.5 × 1010, and 9.5 × 108 particles/mg for nSP30, nSP70, nSP100, mSP300, and mSP1000, respectively), the number of opportunities for contact between the nanosilica particles and coagulation factor XII would have increased with decreasing particle size, thus ultimately leading to the activation of coagulation factor XII. In addition, we need to take into account not only the number of silica particles but also the surface area. The intrinsic cascade pathway involves various factors, such as factor XI and prekallikrein. Therefore, to reveal the mechanism of abnormal activation of the coagulation cascade by nanosilica particles, we need to examine the effects of nanosilica particles on other factors in intrinsic cascade pathways. Increases in the levels of sCD40L and vWF were observed in plasma from the nSP30- and nSP70-treated groups (Figure 6), meaning that nanosilica particles absorbed into the bloodstream induced activation of platelets, which are involved in the activation of coagulation pathways. Although we need to evaluate in greater detail the effects of nanosilica particles on activation or aggregation of platelets, our results and these previous reports suggest that nanosilica particles would induce platelet activation, resulting in activation of an intrinsic cascade pathway. Tavano et al. suggested that synthetic amorphous silica (SAS), which is similar to our silica nanoparticles, and organically modified silica (ORMOSIL) nanoparticles induce significant abnormal activation of the coagulation system via a different mechanism in in vitro studies . More specifically, SAS nanoparticles activate contact coagulation (factor XII dependent) but not TF transcription in monocytes. In contrast, ORMOSIL nanoparticles induce TF-dependent coagulation more efficiently than SAS nanoparticles. The group’s report indicated that the activation of an intrinsic cascade pathway is the main mechanism of amorphous silica nanoparticle–mediated procoagulant activity, thus supporting our study’s conclusions and reiterating the importance of examining the effects of silica nanoparticles on intrinsic coagulation.
In summary, we revealed that intranasally administered nanosilica particles have the potential to induce abnormal activation of a coagulation cascade in mice. Recently, nanosilica particles have been used in food additives and cosmetics, and thus opportunities for such particles to be inhaled by workers during manufacturing are increasing [4, 42]. Furthermore, nanosilica particles are being explored as cancer therapy and drug-delivery agents, and might be administered intranasally in such applications as well [23, 24]. Therefore, the localization and biological effects of intranasally administered nanosilica particles must be elucidated. We expect that further studies of the relationship between localization and biological effects will provide useful information for the development of safer, effective NMs.