2017 年 40 巻 5 号 p. 726-728
We previously reported that unmodified silica nanoparticles with diameters of 70 nm (nSP70) induced liver damage in mice, whereas nSP70 modified with carboxyl or amino groups did not. In addition, we have found that both unmodified and modified nSP70s localize in both Kupffer cells and parenchymal hepatocytes. We therefore evaluated the contributions of nSP70 uptake by these cell populations to liver damage. To this end, we pretreated mice with gadolinium (III) chloride hydrate (GdCl3) to prevent nSP70 uptake by Kupffer cells, subsequently injected the mice with either type of nSP70, and then assessed plasma levels of alanine aminotransferase (ALT). In mice given GdCl3, unmodified nSP70 increased ALT levels. From these data, we hypothesized that in GdCl3-treated mice, the unmodified nSP70 that was prevented from entering Kupffer cells was shunted to parenchymal hepatocytes, where it induced cytotoxicity and increased liver damage. In contrast, GdCl3 pretreatment had no effect on ALT levels in mice injected with surface-modified nSP70s, suggesting that modified nSP70s spared parenchymal hepatocytes and thus induced negligible liver damage. In cytotoxicity analyses, the viability of a parenchymal hepatocyte line was greater when exposed to surface-modified nSP70s than to unmodified nSP70s. These findings imply that the decreased liver damage associated with surface-modified compared with unmodified nSP70 is attributable to decreased cytotoxicity to parenchymal hepatocytes.
Recent progress in nanotechnology has accelerated the development of nanomaterials that are smaller than 100 nm in at least one dimension. Due to their beneficial features, such as increased electrical conductivity and tissue permeability compared with those of microsized materials, nanomaterials have been widely utilized in various fields.1) In particular, silica nanoparticles (nSP), one of the most frequently applied nanomaterials, are used in cosmetics, foods, and medicines.2–4) Therefore, the great potential of nSP to improve our QOL likely will fuel their increased use. However, because of their decreased particle size and associated unique physicochemical properties, nSP are anticipated to exhibit hazards such as acute toxicity and reproductive toxicity.5,6) The continued use of products containing nSP rests on effectively evaluating their safety.
In this context, we have investigated the relationship among the physicochemical properties (particle size and surface characteristics), biodistribution, and biological effects of nSP and have assessed their contributions to the safety of these compounds. We previously reported that nSP (diameter, 70 nm [nSP70]) induced severe liver damage in mice, whereas microsized silica particles (diameter, 300 or 1000 nm) did not.5) In addition, surface-modified nSP70 carrying carboxyl or amino groups (nSP70-C, nSP70-N) prevented hepatic damage in mice,7) despite the presence of similar amounts of unmodified and modified nSP70 in liver.8) These findings suggest that regulating the surface characteristics of nSP is a useful technique to prevent hazard induction. However, precisely how the surface characteristics of nSP influence liver damage is not yet fully understood. In this regard, we have found that, like unmodified nSP70s, surface-modified nSP70s are localized to both non-parenchymal cells (that is, Kupffer cells) and parenchymal hepatocytes.
Here, to identify the hepatic cell population associated with the reduction in liver damage after surface-modification of nSP70, we assessed the liver-associated biological effects due to surface modification of nSP70 in mice.
Silica nanoparticle (diameter, 70 nm) that were either unmodified (nSP70) or surface-modified nSP70-C and nSP70-N were purchased from Micromod Partikeltechnologie (Rostock–Warnemünde, Germany). The particles were sonicated for 5 min and then vortexed for 1 min before use.
AnimalsFemale BALB/c mice were purchased from Nippon SLC (Shizuoka, Japan) and used at 6–8 weeks of age. All animal experimental procedures were performed in accordance with the animal welfare guidelines of Osaka University and the National Institutes of Biomedical Innovation, Health, and Nutrition.
Biochemical AnalysisPlasma levels of alanine aminotransferase (ALT) was measured by using an automated biochemical analyzer (Dri-Chem 7000, FUJIFILM, Tokyo, Japan).
Gadolinium Chloride AssayTo block Kupffer cell uptake of nSP70s in the liver, mice were injected intravenously with gadolinium (III) chloride hydrate (GdCl3) (10 mg/kg) at 30 and 6 h prior to intravenous administration of nSP70 (30 mg/kg), nSP70-C (100 mg/kg), nSP70-N (100 mg/kg), or equal volume of phosphate buffered saline (PBS) (control). Blood samples were collected at 24 h after the injection of nSP70s or PBS.
Cell CultureThe murine hepatocyte line TLR-1 (Institute of Development, Aging, and Cancer; Tohoku University; Japan) was cultured in RITC80–7 medium supplemented with 10 µg/mL transferrin, 1 µg/mL insulin, and 10 ng/mL human Epidermal Growth Factor (Sigma-Aldrich, St. Louis, MO, U.S.A.). The cells were grown in a humidified incubator at 33°C (95% room air, 5% CO2).
Cytotoxicity AssayThe cytotoxicities of nSP70s against TLR-1 cells were evaluated by measuring the release of lactate dehydrogenase. Cells (1×104 cells per well in 96-well plates) were cultured with nSP70, nSP70-C, or nSP70-N at 10, 30, 90, 270, or 810 µg/mL for 24 h. The lactate dehydrogenase activity of the culture supernatant was determined by using a commercial cytotoxicity test (Wako Pure Chemical Industries, Ltd., Osaka, Japan) according to the manufacturer’s instructions.
Statistical AnalysesThe results are expressed as means±standard error (S.E.) or standard deviation (S.D.) Differences were compared by using Bonferroni tests after one-way ANOVA.
As mentioned in the Introduction, we have found that both unmodified and modified nSP70s are localized in both Kupffer cells and parenchymal hepatocytes. Therefore, to identify the hepatic cell population associated with the reduction in liver damage after surface-modification of nSP70, we used GdCl3 to prevent nSP70s from entering Kupffer cells.9) Among mice treated with unmodified nSP70, the plasma ALT levels of GdCl3-treated mice were significantly higher than those of GdCl3-untreated mice (Fig. 1). These data suggest that, in the current study, Kupffer cells were protective against the liver damage caused by unmodified nSP70, although other authors report that Kupffer cells are involved in worsening or improving liver damage.10) Moreover, the increased ALT level after inhibiting Kupffer cells suggests that GdCl3 pretreatment increased the amount of unmodified nSP70 transported into the parenchymal hepatocytes of mice, thus injuring those cells and increasing liver damage; however this hypothesis requires further quantitative analysis. In contrast, among the groups injected with surface-modified nSP70s, the plasma levels of ALT were similar between GdCl3-treated and -untreated mice (Fig. 1). These data suggest that the parenchymal hepatocytes of mice treated with surface-modified nSP70 contributed little to the amount of hepatic injury induced.
Mice were pretreated with GdCl3, an inhibitor of uptake by Kupffer cells, and then intravenously injected with unmodified or surface-modified nSP70s. Plasma levels of ALT were analyzed as an indicator of liver damage. The results are expressed as mean±S.E. (n=3 or 4; * p<0.01 compared with value for PBS-treated group; ** p<0.01 compared with value for nSP70-treated group; differences compared by one-way ANOVA with Bonferroni tests).
To estimate the cytotoxicity of unmodified or surface-modified nSP70s on parenchymal hepatocytes, we examined the effects of the various nSP70s on the release of lactate dehydrogenase from cultures of TLR-1 cells, a murine hepatocyte line. According to the amount of lactate dehydrogenase in the culture supernatant, treatment with unmodified nSP70 dose-dependently decreased cell viability, whereas exposure to neither nSP70-C nor nSP70-N group influenced viability (Fig. 2). These data suggest that the cytotoxicity-sparing effect due to surface modification of nSP70s on parenchymal hepatocytes reduced the liver damage in the nSP70-C- and nSP70-N-treated groups. In addition, the viability of parenchymal hepatocytes isolated from liver tissue tended to decrease in samples from mice injected intravenously with unmodified nSP70 compared with nSP70-C or nSP70-N in our preliminary in vivo examination.
We previously showed that the generation of reactive oxygen species (ROS) by several mammalian cell lines was significantly lower in cultures treated with nSP70-C or nSP70-N than in those exposed to unmodified nSP70.11) Therefore, we speculate that a difference in the level of ROS generated in the cells is one of the factors contributing to the differing cytotoxicities of various nSP70 formulations on parenchymal hepatocytes.
Cells of the murine hepatocyte line TLR-1 were incubated with the indicated concentrations of nSP70 (circles), nSP70-C (squares), or nSP70-N (triangles) for 24 h. The cytotoxicity of the nSP70s against TLR-1 cells was evaluated by measuring the concentration of lactate dehydrogenase released to the culture supernatants. Data are presented as mean ±1 S.D. (n=3; * p<0.01 [one-way ANOVA with Bonferroni test] compared with value for mice treated with unmodified nSP70s).
Furthermore, it has recently been reported that nanomaterials become coated with various protein from biological fluids (a so-called ‘protein corona’) once they are in the body and that the protein corona alters the biological responses to nanoparticles.12) For example, the induction of ROS in human acute monocytic leukemia (THP-1) macrophages decreased when Fe3O4 nanoparticles were encased in a protein corona.13) In addition, we previously demonstrated that coagulation factor XII bound more extensively to the surface of nSP70 than to nSP70-N by using Western blot analysis.8) Therefore, because differences in the surface characteristics of nanomaterials influence the protein profile of the corona,14) differences in the amount or species of the proteins bound by unmodified compared with surface-modified nSP70s may influence ROS generation.
In conclusion, the suppression of liver damage due to surface modification of nSP70s is attributable to their reduced cytotoxicity to parenchymal hepatocytes. Defining the mechanism underlying the hepatotoxicity of nanomaterials will facilitate the creation of safer products.
This study was supported, in part, by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and from the Japan Society for the Promotion of Science (JSPS); by a Grant-in-Aid for JSPS Fellows; by Health Labour Sciences Research Grants from the Ministry of Health, Labour and Welfare of Japan; by The Takeda Science Foundation; by The Research Foundation for Pharmaceutical Sciences.
The authors declare no conflict of interest.
