Silicon dioxide (SiO 2 ) nanoparticles (NPs) have been widely used in the biomedical field, such as in drug delivery and gene therapy. However, little is known about the biological effects and potential hazards of SiO 2 . Herein, the colloidal SiO 2 NPs with two different sizes (20 nm and 100 nm) and different charges (L-arginine modified: SiO 2 EN20[R] , SiO 2 EN100[R] ; and negative: SiO 2 EN20[&#;] , SiO 2 EN100[&#;] were orally administered (750 mg/kg/day) in female C57BL/6 mice for 14 days. Assessments of immunotoxicity include hematology profiling, reactive oxygen species generation and their antioxidant effect, stimulation assays for B- and T-lymphocytes, the activity of natural killer (NK) cells, and cytokine profiling. In vitro toxicity was also investigated in the RAW 264.7 cell line. When the cellularity of mouse spleen was evaluated, there was an overall decrease in the proliferation of B- and T-cells for all the groups fed with SiO 2 NPs. Specifically, the SiO 2 EN20(&#;) NPs showed the most pronounced reduction. In addition, the nitric oxide production and NK cell activity in SiO 2 NP-fed mice were significantly suppressed. Moreover, there was a decrease in the serum concentration of inflammatory cytokines such as interleukin (IL)-1β, IL-12 (p70), IL-6, tumor necrosis factor-α, and interferon-γ. To elucidate the cytotoxicity mechanism of SiO 2 in vivo, an in vitro study using the RAW 264.7 cell line was performed. Both the size and charge of SiO 2 using murine macrophage RAW 264.7 cells decreased cell viability dose-dependently. Collectively, our data indicate that different sized and charged SiO 2 NPs would cause differential immunotoxicity. Interestingly, the small-sized and negatively charged SiO 2 NPs showed the most potent in vivo immunotoxicity by way of suppressing the proliferation of lymphocytes, depressing the killing activity of NK cells, and decreasing proinflammatory cytokine production, thus leading to immunosuppression.

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In this study, we investigated the potential immunotoxicity of colloidal silicon dioxide (SiO 2 ) NPs with two different sizes (20 nm and 100 nm) and different charges (L-arginine modified: SiO 2 EN20[R] , SiO 2 EN100[R] ; and negative: SiO 2 EN20[&#;] , SiO 2 EN100[&#;] ) in mice and in the RAW 264.7 cell line. Accordingly, cytotoxicity was performed in vitro using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Rockville, MD, USA). The primary indicators of immune toxicity were also assessed in vivo, such as body weight measurement and hematology profiles. The NP-induced oxidative effect was also examined with reactive oxygen species (ROS) generation, superoxide dismutase (SOD) activity, and intracellular levels of glutathione peroxidase (GPx). The cellularity of the spleen and analysis of the functional capacity of specific immune cells were evaluated by stimulation assays for B- and T-lymphocytes, and the activity of natural killer (NK) cells was examined. In addition, we also focused on the inflammatory responses induced by NPs; therefore, the concentration of cytokines was determined.

The growing fabrication and characterization of silica nanoscale materials has received attention in biomedical research, such as in the development of biosensors, enzyme immobilization, controlled drug release and delivery, and cellular uptake. 1 &#; 6 Since these various studies on nanomedicine have emerged, the medical applications of nanoparticles (NPs) were highlighted, especially for the development of anticancer agents. Mesoporous silica NPs (MSNs) attracted great attention in the last few decades for their wider plausible application in the emerging field of nanomedicine. MSNs are inorganic nanocarriers that are known to be highly stable in a physicochemical and biochemical context; thus, they are vitally important in the construction of anticancer medicine. 6 , 7 Recently, these MSNs received United States Food and Drug Administration approval as inorganic carriers in nanomedicine, and they are considered one of the most promising inorganic nanobiomaterials. 7 , 8 Their potential applications seem endless based on the unique physicochemical characteristics of this nanomaterial, such as varying sizes, shapes, chemical composition, and assembly. 9 Meantime, the special physicochemical characteristics of silica posed concerns about their potential environmental and health implications. 10 To date, animal exposure to colloidal silica confirmed liver damage 11 and moderate to severe pulmonary inflammation and tissue damage, primarily induced by oxidative stress and apoptosis. 12 , 13 The physicochemical properties play an important role in the toxic reaction of silica NPs. Small particles mean that there is a larger surface area, and this might indicate an increase in surface reactivity, which enables the NPs to interact with cell biomolecules. 9 The altered surface charge of NPs provides a unique way to facilitate their uptake into the interior structure of the cells. 14 However, most studies have focused on pulmonary and liver toxicity; very few studies have overlooked the toxicological effects of silica NPs on the immune response in vivo. 15 &#; 17 In addition, the influence of NP properties (for example, size, surface charge) on their potential hazards to the biological system needs to be elucidated.

Serum levels of selected cytokines, such as interleukin (IL)-1β, IL-6, IL-10, IL-12p70, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ, were measured using a Luminex bead-based suspension array system (Bio-Rad Laboratories, Hercules, CA, USA). This Luminex-based multiplexed system combines the principle of a sandwich immunoassay with fluorescent bead-based technology. Briefly, each set of premixed beads coated with the target antibodies was added to the well, and it was incubated with the sample in a 96-well round-bottomed microtiter plate to react with specific analytes. Then, premixed detection antibodies were added to the wells followed by a fluorescently labeled reporter molecule that specifically binds the analyte. Standard curves for each cytokine were generated using the standard control concentrations provided in the kit. Each step requires a specific incubation time, with shaking at room temperature and washing steps. All washes were performed using a Bio-Plex ® Pro wash station (Bio-Rad Laboratories). Finally, the samples were then read using the Bio-Plex ® suspension array reader, and the raw fluorescence data were analyzed using the Bio-Plex ® Manager&#; software using five-parameter logistic fitting (Bio-Rad Laboratories).

The murine lymphoma cancer cell line, YAC-1 (American Type Culture Collection), as target cells were cocultured with NK-enriched murine splenocytes. The percentage of target cells killed by effector NK cells was determined. Briefly, various effector cell dilutions were prepared: 1×10 5 cells/well for 100:1; 5×10 4 cells/well for 50:1; and 2.5×10 4 cells/well for 25:1. The target cells (1×10 3 cells/well) were then cocultured with the different effector cell dilutions prepared in a 96-well plate and incubated for 6 hours at 37°C in a humidified incubator with 5% CO 2 . The CCK-8 was conducted according to the manufacturer&#;s instructions. A special highly water soluble tetrazolium salt (WST-8) (2-[2-methoxy-4-nitrophenyl]-3[4-nitrophenyl]-5[2-4disulfophenyl]-2H-tetrazolium, monosodium salt) was added to the culture. WST-8 is reduced by dehydrogenase activities in cells to give the orange formazan dye, which is soluble in the culture media. The amount of the formazan dye generated by dehydrogenase in cells is directly proportional to the number of living cells. After 1 hour of incubation with the WST-8 solution, the cell suspension was then colorimetrically measured at 450 nm by a DTX-880 multimode microplate reader, and the number of live cells at the different ratios was determined.

For the isolation of splenocytes, spleens were aseptically removed from recently sacrificed mice and the tissue was transferred to a tube containing 1× PBS on ice. Splenocyte suspensions were prepared by blandly squeezing the spleen between the frosted ends of the two sterile microscope slides into a 100 mm tissue culture grade Petri dish. The slides were rinsed at regular intervals with 1× PBS. The cells were formed into a single suspension using a pipette. After that, the cell suspensions were filtered through a cell strainer. Next, the cell suspensions were centrifuged at 1,500 rpm at 4°C for 5 minutes to produce pellets. For the optimal lysis of erythrocytes, the pellets were resuspended in 5 mL of red blood cell lysis buffer and incubated on ice for 5 minutes with occasional shaking. The reaction was stopped by diluting the lysis buffer with 25 mL of 1× PBS. Thereafter, cells were spun (1,500 rpm at 4°C for 5 minutes), and the supernatant was carefully removed. The pellet was then washed two times in 1× PBS and resuspended in Roswell Park Memorial Institute (RPMI)- supplemented with 3% FBS and 1% antibiotic&#;antimycotic. Cells were then counted. The viability of the cells used in all the experiments was higher than 95%, as measured by the trypan blue exclusion method (Sigma-Aldrich, St Louis, MO, USA).

Discussion

There is an almost unanimous opinion about the potential toxicity of NP exposure, and it includes ROS generation, proinflammatory responses, and cell death.25,26 However, most of the studies on NPs have focused on lung and liver toxicity, such that the toxic effect of NPs on the immune system is poorly documented.15,16 In addition, the influence of NP size and electrostatic charge on potential immunotoxicity remains to be elucidated. The present study addressed the immunotoxicity of different sizes and electrostatic charges of SiO2 NPs that were fed to mice for 14 days (orally administered at doses of 750 mg/kg/day). In particular, our study shows that immune dysfunction from exposure to the varying sizes and electrostatic charges of SiO2 would lead to immunosuppression. This was evidenced by suppressing the proliferation of lymphocytes, by depressing the killing activity of NK cells, and by decreasing inflammatory cytokine production. In addition, our data showed that different sizes and charges of SiO2 NPs could lead to differential immunotoxicity in vivo. To clarify this, we first investigated the immune function in mouse spleen lymphocytes. Mitogens such as Con-A and LPS are used to stimulate T-cells and B-cells, respectively, and to assess immune function. We found that the proliferations of B- and T-cells were reduced when mice were fed with SiO2 NPs (SiO2EN20[R], SiO2EN20[&#;]2, and SiO2EN100[&#;]). This is parallel to the decreased lymphocyte count in the blood, as shown in and . Our results are similar to the findings of Lee et al,27 who demonstrated that the intraperitoneally administered 100 nm colloidal silica NPs showed no increase in their proliferative responses to the lymphocyte mitogens. Since lymphocytes play pivotal roles in the immune response, altered B- and T-cell proliferations might culminate in the dysregulation of the immune response.28 This result might suggest that SiO2 NPs, specifically the groups fed negatively charged NPs, may show a decrease in the white pulp component of the spleen, as there is a significant decrease in lymphocyte count.

Cumulative data showed that oxidative stress is another toxic mechanism of NPs.17,29 Oxidative stress and NO are closely linked to inflammatory responses. NO is implicated in phagocytosis, as well as in the pathogenesis of inflammation.30,37 The impaired production of NO leads to undesired effects, such as inflammation and tissue damage.30 Thus, the abnormal production of NO, such as increases or decreases upon exposure to silica NPs, may induce a proinflammatory response.30 ROS is known to stimulate NO production. Of note, there is the decreased generation of ROS in negatively charged SiO2EN20(&#;) and SiO2EN100(&#;)-fed mice; therefore, there was decreased NO generation in these given groups. Further, increased production of ROS in SiO2EN20(R)- and SiO2EN100(R)-fed mice led to increased NO generation, as compared to the SiO2EN20(&#;)- and SiO2EN100(&#;)-fed groups. The NP surface charge influenced the capacity of ROS production.31,32 L-arginine-coated SiO2 NPs are capable of inducing intracellular ROS; however, the decreased ROS production among the mice that were fed negatively charged SiO2 NPs might be due to earlier or later ROS production that occurred in these groups. In addition, the decreased ROS generation among mice fed negatively charged SiO2EN20(&#;) and SiO2EN100(&#;) may be due to the leakage of the fluorescent product from the cell due to membrane damage, as there is a higher tendency of cytotoxicity on negative charge, as shown by in vitro viability. Size and charge influence the cytotoxicity of engineered nanomaterials.25,32 When comparing the cytotoxicity of particle size and surface charge, we found that negatively charged NPs tended to be higher in toxicity, and 20 nm NPs were the most toxic in a murine macrophage cell line (RAW 264.7) (see ). In line with our results, the reported data showed that phagocytic cells preferentially interacted with negatively charged particles.33 Therefore, the higher toxicity of the negatively charged SiO2EN20(&#;) is due, in part, to the stronger interaction with the macrophage cells. Given this, the in vitro cytotoxicity and in vivo ROS production showed that negatively charged SiO2 NPs are more toxic than their negative counterparts. To further prove our findings, we selected other immunotoxicity parameters, such as cytokine profiling and NK cell killing activity.

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Any invading pathogens can trigger an inflammatory response, which involves the secretion of inflammation-induced mediators such as cytokines.34 In an inflammatory response, activated immune cells recognize the NPs by their unique physicochemical characteristics, such as their surface charge and surface properties, thus inducing the cytokines to attract more cells to eradicate the NPs.20 Electrostatic charges of the NPs are important parameters in an inflammatory response. As noted by Tan et al,35 cationic (positively charged) engineered nanomaterials, such as liposomes, induced the secretion of cytokines, such as TNF, IL-12, and IFN-γ. In relation to this, mice fed L-arginine surface modified NPs (SiO2EN20[R] and SiO2EN100[R]) showed induced secretion of cytokines such as IL-12, IFN-γ, and TNF-α, as compared to the anionic NP (negatively charged SiO2EN20[&#;] and SiO2EN100[&#;])-fed mice. Consistent with these results, the SiO2EN20(R)-fed group showed increased WBC production, as compared to the negatively charged (SiO2EN20[&#;] and SiO2EN100[&#;])-fed mice (which are both in reference to their corresponding normal control group WBC levels). With respect to the relationship between the WBC level and each type of WBC (such as lymphocytes and monocytes) with inflammation, activated WBCs trigger the secretion of cytokines.36 Schwentker et al37 reported that NO could affect the expression and activity of cytokines. Further, particle size and surface area are also important parameters that affect in vivo bioreactivity.38 NPs are predictive in stimulating cytokine production, specifically the ultrafine particles.39 Our study showed that induction of cytokine production is clearly seen in SiO2EN20(R)-fed mice. NO production was low in negatively charged SiO2EN20(&#;)-fed mice. In parallel, the secretion of cytokines (IL-1β, TNF-α, IL-12p70, and IFN-γ) in negatively charged SiO2EN20(&#;) and SiO2EN100(&#;)-fed mice was repressed. Of these cytokines, IL-12p70 (SiO2EN20[&#;], SiO2EN100[&#;]) and TNF-α (SiO2EN100[&#;]) were significantly decreased in concentration, as compared to the levels found among the SiO2EN20(R) and SiO2EN100(R)-fed mice and among the mice in the NC group. Negatively charged SiO2EN20(&#;)-fed mice showed the least secretion of IFN-γ among all of the groups. These two cytokines (IL-12p70 and IFN-γ) are involved in the activation of NK cells, which were found in decreased concentrations in mice fed with negatively charged SiO2EN20(&#;) NPs. In addition, the decreased proliferation of B-cells in negatively charged SiO2EN20(&#;)-fed mice also supports our findings, where B-cells also function to secrete cytokines such as IL-1, IL-10, IL-6, IFN-γ, and TNF-α which, in turn, activates the antigen-presenting cells.40 Physicochemical properties such as the size and charge of NPs determine their immunotoxicity, and they may also enhance their biological reactivity.41 The biological activity of NPs increases as the particle size decreases.42 However, this trend can be altered by a small change in the particle&#;s surface charge. NPs with positively charged surfaces could be more easily up-taken due to the attractive interaction to the negative cell membrane.43 Despite using varying sizes and electrostatic charges of NPs in this study, other factors need to be considered as well, such as the different exposure periods (chronic or acute) and the different dosages (low, medium, and high) of the NPs, in order to fully discuss, in detail, the potential immunotoxicity of SiO2. In addition, our study focused on the spleen, as it was suggested by other studies that the spleen is one of the major target organs for toxicity.44,45 Moreover, the spleen is also involved in the initiation of immune responses, such that lymphocyte proliferation make take place in this given organ.27 However, to fully elucidate the biosafety of SiO2 NPs in particular, the immune system and other target organs, such as the lymph nodes and liver, need to be tested. While this paper provides evidence on SiO2 immunotoxicity, the underlying mechanism still needs to be elucidated.

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Silicon Dioxide (SiO2) Nanopowder/Nanoparticles

S-type, Spherical, Purity: 99.95+%, Size: 13-22 nm, Nonporous and Amorphous 

Technical Properties:

2

/g)   

Applications: 

Silicon dioxide nanoparticle is used as additive for plastics, rubber, ceramics, porcelain, glass, adhesives, fibers, and many other products.
It is added to concrete and construction composites as strengthening filler. It has also applications in biomedical field such as drug delivery
and theranostics. Silicon dioxide is also used in environmental protection products.

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Immunotoxicity of silicon dioxide nanoparticlessilicon dioxide nanoparticles with ...

Silicon Dioxide (SiO2) Nanopowder/Nanoparticles