Effects of Metal Oxide Nanoparticles on Toll-Like Receptor mRNAs in Human Monocytes

For the widespread application of nanotechnology in biomedicine, it is necessary to obtain information about their safety. A critical problem is presented by the host immune responses to nanomaterials. It is assumed that the innate immune system plays a crucial role in the interaction of nanomaterials with the host organism. However, there are only fragmented data on the activation of innate immune system factors, such as toll-like receptors (TLRs), by some nanoparticles (NPs). In this study, we investigated TLRs’ activation by clinically relevant and promising NPs, such as Fe3O4, TiO2, ZnO, CuO, Ag2O, and AlOOH. Cytotoxicity and effects on innate immunity factors were studied in THP-1(Tohoku Hospital Pediatrics-1) cell culture. NPs caused an increase of TLR-4 and -6 expression, which was comparable with the LPS-induced level. This suggests that the studied NPs can stimulate the innate immune system response inside the host. The data obtained should be taken into account in future research and to create safe-by-design biomedical nanomaterials.

In particular, the iron oxide NPs, Fe 3 O 4 (magnetite), are MRI contrast agents used for the imaging of cancer, and of cardiovascular and inflammatory diseases [14,15]. In addition, magnetite has been approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of iron deficiency anemia [16]. Titanium dioxide (TiO 2 ) NPs are used for cleaning air and water, and are used in pigments, cosmetics, and skincare products [17]. Zinc oxide (ZnO) NPs are among the most commonly used nanomaterials, with a wide range of applications including biomedical imaging, drug and gene delivery, biosensing, and antibacterial and antifungal applications [18]. Copper oxide (CuO) has a high potential in the development of biosensors [19][20][21], antifouling coatings [22,23], and biocidal [24][25][26] and antitumor agents [27][28][29]. Boehmite (AlOOH) can be used as an adjuvant for vaccines [30,31]. Silver oxide (Ag 2 O) NPs have the potential for diagnostic biological probes [32], and antibacterial and anticancer agents [33][34][35].

Characterization Techniques
The crystal phase and crystallinity of samples were studied by the X-ray diffraction (XRD) method (Rigaku SmartLab 3 diffractometer (Tokyo, Japan) of the engineering center of the Saint Petersburg State Technological Institute (Technical University)) using Cu-Kα irradiation (λ = 1.54 Å). Samples were scanned along 2θ in the range of 10-70 • at 0.5 degrees/min. For XRD analysis, samples were dried at 120 • C for 4 h. For SEM analysis, the samples were dried in vacuo for 2 h and examined using a Tescan VEGA 3 scanning electron microscope (Brno, Czech Republic). The particle size and zeta potential in colloidal solutions were measured using a Photocor EPM/Photocor Compact Z (Moscow, Russia). The surface area, pore volume, and pore size distribution were investigated using Quantachrome Nova 1200e (Boynton Beach, FL, USA) by nitrogen adsorption at 77 K, and analyzed using the BET and BJH equations. Prior to analysis, all samples were degassed at 110 • C for 4 h.

MTT Assay
To evaluate the NPs' cytotoxicity, the human monocytic THP-1 cells were maintained in RPMI 1640 (Biolot, Saint-Petersburg, Russia) supplemented with 10% fetal bovine serum (Gibco, Australia) and 50 µg/mL gentamycin (Biolot Saint-Petersburg, Russia) at 37 • C, 5% CO 2 . Cells at a logarithmic phase of growth were plated (5 × 10 3 /well) into 96-well plates and treated for 72 h with NPs resuspended directly in the culture medium to reach final concentrations of 0.23-30 µg/mL. The volume of added NPs from the stock in water was <5% of the total volume of the culture medium in the wells. After the completion of cell exposure, 20 µL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 0.5 mg/mL) solution in saline buffer were added to each well for 1.5 h. Then the solution was aspirated, and formazan granules were dissolved in 200 µL of dimethyl sulfoxide. Optical density was measured at 570 nm on a Tecan Infinite 50 spectrophotometer (Mennedorf, Switzerland). Cell viability was calculated as the percentage of optical densities in wells, with each concentration of NPs normalized to the optical density of untreated cells (100%).

NPs' Influence on TLR Gene Expression
Cells at the logarithmic phase of growth were plated (10 6 /well) into 6-well plates and treated for 24 h with NPs resuspended directly in the culture medium at final concentrations (according to the MTT tests) in which cell viability was >80%: for Fe 3 O 4 and AlOOH, 30 µg/mL; for TiO 2 , 25 µg/mL; for Ag 2 O, 15 µg/mL; for CuO, 0.5 µg/mL; and for ZnO, 1 µg/mL. As a positive control for TLR signaling activation, we used lipopolysaccharide (LPS) (Sigma, St. Louis, Missouri, USA).

RNA Extraction, Reverse Transcription and Quantitative PCR
An RNA extraction kit ExtractRNA (Evrogen) was used to extract total RNA from THP-1 cells. Concentration and purity of total RNA obtained after the extraction were quantified based on the absorbance at 260 nm using a Nanophotometer (Implen, Munich, Germany). Reverse transcription was performed using an MMLV-RT kit containing reverse transcriptase (Evrogen, Moscow, Russia) with the addition of hexamer primers to obtain cDNA from the RNA template. Both total RNA extraction and reverse transcription were performed according to the manufacturer's protocols. Quantitative real-time PCR with the fluorescent probe SYBR-Green was used to assess the TLRs' mRNAs in response to NPs or LPS. To normalize the expression data, we used the housekeeping reference gene beta-2-microglobulin. PCR was performed using the qPCRmix-HS SYBR reagent kit (Evrogen Moscow, Russia). Primers were the following: TLR-4 Forward: GCTCTGCCTTCACTACAGGGACT, Reverse: CTGGGACACCACGACAATAACC; TLR-6 Forward: TGGGCTAACATTAGAGCCGC, Reverse: GGCATGAGGATAATGGAGGCA; beta-2-microglobulin Forward: GATGAGTATGCCTGCCGTGT, Reverse: TGCGGCATCTTCAAACCTCC. Primer sequences were selected using NCBI Primer-BLAST and synthesized in Evrogen. Real-time PCR was performed on a CFX Connect™ Real-Time PCR Detection System (BioRad Laboratories, Hercules, CA, USA). Relative quantification of gene expression was performed using the comparative Cq method of calculating threshold cycles of genes of interest. Relative gene expression data were normalized to an internal control [59]. The standard deviation was determined based on values from triplicate samples.

Statistics
Each experiment in mammalian cultures was carried out in triplicate. Data are presented as mean ± SD. Statistical analyses were performed using the Student's t-test or Mann−Whitney test (Statistica 6, StatSoft Inc., Tulsa, OK, USA). Statistical significance was considered at p < 0.05. The surface of metal oxides by low-temperature nitrogen physisorption was studied (Table 1 and Figure 3). All newly synthesized materials were mesoporous with a narrow pore size distribution. The surface area calculated by BET was the largest for TiO2, Fe3O4, and AlOOH NPs (167, 120, and 170 m 2 /g, respectively), which is consistent with the initial sizes of the obtained particles.  The described procedures allowed us to obtain the hydrosols of metal NPs with various parameters. Determining the modality of hydrosols by dynamic light scattering (DLS), we found that the hydrosols of zinc and copper were characterized by a multimodal distribution ( Figure 1B). This meant that these hydrosols were less stable compared to the sols of iron, titanium, and alumina oxides. In particular, the hydrodynamic diameter of the resulting particles was 40-600 nm. The reduced physical stability of the hydrosols of zinc, copper, and silver oxides can also be explained by the relatively low value of zeta potential (up to 15 mV modulo). Table 1 presents the values of zeta potential for the studied systems. One may see that the charge of the particles of iron, titanium, copper, zinc, and aluminum is positive, whereas silver NPs are negatively charged (ζ = −15 mV). It is worth noting that the charge does not affect the stability of aqueous solutions. The structures of NPs were also analyzed using scanning electron microscopy (SEM; Figure 2). The particle size distribution was determined according to SEM. The samples of CuO were rods, which explained their large size and multimodal particle size distribution according to DLS. Other new NPs had a spherical shape, while their distribution was quite narrow. A comparison of size distributions by SEM for Ag 2 O and ZnO with DLS particle distribution demonstrated that the measured results are inconsistent. This fact can be related to particle agglomeration into large clusters.

Characterization of NPs
The surface of metal oxides by low-temperature nitrogen physisorption was studied (Table 1 and Figure 3). All newly synthesized materials were mesoporous with a narrow pore size distribution. The surface area calculated by BET was the largest for TiO 2 , Fe 3 O 4 , and AlOOH NPs (167, 120, and 170 m 2 /g, respectively), which is consistent with the initial sizes of the obtained particles.

Cytotoxicity of NPs
Fe3O4, TiO2, and AlOOH NPs evoked no cytotoxic effects on human monocytic cells (THP-1 line) at the range of concentrations of 0.234-30 μg/mL for at least 72 h of continuous exposure. The cell survival rate did not fall below 80%. No morphological signs of death were detectable, indicating that even the maximum concentrations used in this experiment were tolerable. Ag2O NPs were cytotoxic at 30 μg/mL, whereas at 0.234-15 μg/mL, cell viability was ≥80%. CuO and ZnO were toxic at ≥15

Fe 3 O 4 , TiO 2 , and AlOOH NPs evoked no cytotoxic effects on human monocytic cells (THP-1 line)
at the range of concentrations of 0.234-30 µg/mL for at least 72 h of continuous exposure. The cell survival rate did not fall below 80%. No morphological signs of death were detectable, indicating that even the maximum concentrations used in this experiment were tolerable. Ag 2 O NPs were cytotoxic at 30 µg/mL, whereas at 0.234-15 µg/mL, cell viability was ≥80%. CuO and ZnO were toxic at ≥15 µg/mL (Figure 4). In general, the results of the cytotoxicity analysis coincide with the previously published results [61,62]. Using the results of this analysis, we have calculated the optimal NP concentrations to analyze their effects on TLR-4 and -6 expression.

Cytotoxicity of NPs
Fe3O4, TiO2, and AlOOH NPs evoked no cytotoxic effects on human monocytic cells (THP-1 line) at the range of concentrations of 0.234-30 μg/mL for at least 72 h of continuous exposure. The cell survival rate did not fall below 80%. No morphological signs of death were detectable, indicating that even the maximum concentrations used in this experiment were tolerable. Ag2O NPs were cytotoxic at 30 μg/mL, whereas at 0.234-15 μg/mL, cell viability was ≥80%. CuO and ZnO were toxic at ≥15 μg/mL (Figure 4). In general, the results of the cytotoxicity analysis coincide with the previously published results [61,62]. Using the results of this analysis, we have calculated the optimal NP concentrations to analyze their effects on TLR-4 and -6 expression.

NPs Enhance TLR-4 and TLR-6 mRNA Levels in THP-1 Cells
In this study, we aimed to demonstrate the immune system response caused by the exposure to NPs on the model of activation of TLRs in THP-1 monocytes. This study is relevant because the NPs used in the investigation have a high therapeutic potential and can be used to create new approaches to the treatment of diseases, such as bacterial and fungal infections, through the additional stimulation of the immune system [36][37][38]. TLRs are present in most cell types and are part of the

NPs Enhance TLR-4 and TLR-6 mRNA Levels in THP-1 Cells
In this study, we aimed to demonstrate the immune system response caused by the exposure to NPs on the model of activation of TLRs in THP-1 monocytes. This study is relevant because the NPs used in the investigation have a high therapeutic potential and can be used to create new approaches to the treatment of diseases, such as bacterial and fungal infections, through the additional stimulation of the immune system [36][37][38]. TLRs are present in most cell types and are part of the signaling pathways that respond to various compounds; in particular, bacterial cell wall components such as lipopolysaccharide [39,40]. They cause the activation of cellular mechanisms that lead to increased activity of T and B cells, as well as macrophages. This provides an immune response [62].
To estimate the influence of NPs on TLR-4 and -6 mRNAs in THP-1 monocytes, we used the concentrations at which cell viability was at least 80% as determined in MTT tests. We accepted that the corresponding equipotent values for these viability levels were as follows: 30 µg/mL for Fe 3 O 4 and AlOOH, 25 µg/mL for TiO 2 , 15 µg/mL for Ag 2 O, 1 µg/mL ZnO, and 0.5 µg/mL for CuO. Here, we incubated cells with these NPs or with 1µg/mL LPS as a positive inductor of TLR response for 24 h.
All the studied NPs increased the TLRs' expression to different degrees ( Figure 5), however, the maximum induction was comparable with one after the LPS exposure. The maximum induction of TLR-4 was observed under the AlOOH NPs' influence, which increased the expression 1.5-fold. Of note, the level of TLR-4 following AlOOH was comparable with that of LPS. CuO, ZnO, and TiO 2 NPs increased the expression less significantly: 1.2, 1.2, and 1.1-fold, respectively. Ag 2 O and Fe 3 O 4 did not affect the TLR-4 expression. TLR-6 was also the most induced by AlOOH NPs-1.6-fold. Among the other particles, Ag 2 O, TiO 2 , and CuO were more potent: fold induction values were 1.5, Nanomaterials 2020, 10, 127 8 of 12 1.5, and 1.5, respectively. The least comparative effects were produced by ZnO and Fe 3 O 4 NPs, which increased the expression 1.4-fold. Thus, the most potent TLR inductor is AlOOH NPs. CuO and TiO 2 NPs also stimulated the expression of both TLRs, but to a lesser extent. Silver did not affect the TLR-4 but instead strongly induced TLR-6. The most unreactive NPs were Fe 3 O 4 . Nanomaterials 2020, 10, x; doi: FOR PEER REVIEW www.mdpi.com/journal/nanomaterials For some NPs we studied, immune response stimulation, including TLR-mediated stimulation, has been shown earlier [41][42][43][44][45][46][47][48][49]. In some cases, the TLR expression data we obtained were slightly lower than the previously published data. In particular, it has been shown that magnetite NPs specifically induced macrophage autophagy through activation of TLR-4 [16]. In our case, the Fe3O4 NPs did not affect the TLR-4. In addition, it has been shown that among the TLRs, TLR-6 was the most potent activator of inflammatory reactions induced by ZnO NPs [54]. In our study, they did activate the TLR-6 more strongly than the TLR-4, but in comparison with other NPs, their effect on the TLR-6 was one of the lowest. Such a discrepancy may be due to the peculiarities of NP synthesis, which causes their increased toxicity and immunogenicity, as well as the peculiarities of experiments on biological models. In this regard, it is especially important to conduct a systematic study of a set of NPs characterized and synthesized simultaneously in the same or similar conditions.

Conclusions
In this study, we analyzed the influence of metal oxide NPs on innate immunity by testing TLR-4 and -6 mRNAs in response to these nanomaterials in the human monocyte cell line. We detected no cytotoxicity for 72 h at 30 μg/mL for Fe3O4 and AlOOH, 25 μg/mL for TiO2, 15 μg/mL for Ag2O, 0.5 For some NPs we studied, immune response stimulation, including TLR-mediated stimulation, has been shown earlier [41][42][43][44][45][46][47][48][49]. In some cases, the TLR expression data we obtained were slightly lower than the previously published data. In particular, it has been shown that magnetite NPs specifically induced macrophage autophagy through activation of TLR-4 [16]. In our case, the Fe 3 O 4 NPs did not affect the TLR-4. In addition, it has been shown that among the TLRs, TLR-6 was the most potent activator of inflammatory reactions induced by ZnO NPs [54]. In our study, they did activate the TLR-6 more strongly than the TLR-4, but in comparison with other NPs, their effect on the TLR-6 was one of the lowest. Such a discrepancy may be due to the peculiarities of NP synthesis, which causes their increased toxicity and immunogenicity, as well as the peculiarities of experiments on biological models. In this regard, it is especially important to conduct a systematic study of a set of NPs characterized and synthesized simultaneously in the same or similar conditions.

Conclusions
In this study, we analyzed the influence of metal oxide NPs on innate immunity by testing TLR-4 and -6 mRNAs in response to these nanomaterials in the human monocyte cell line. We detected no cytotoxicity for 72 h at 30 µg/mL for Fe 3 O 4 and AlOOH, 25 µg/mL for TiO 2 , 15 µg/mL for Ag 2 O, 0.5 µg/mL for CuO, and 1 µg/mL for ZnO (the range of investigated concentrations was chosen according to the maximum NP concentration, forming stable sol in aqueous solution). All studied NPs activated TLR-6 expression, whereas AlOOH enhanced both TLR-4 and -6. Thus, the use of these NPs in vivo may have a dual effect, due to stimulation of the innate immune system. The effect may be beneficial due to the increased expression of anti-inflammatory cytokines. It is of particular importance for drug development against bacterial or fungal infections, where additional stimulation of the immune system can accelerate the antimicrobial response and tissue repair. However, these effects should be kept in mind when using these materials for anticancer drug development, since the attraction of immune cells to the tumor may be clinically unfavorable. At any rate, it should be noted that the maximum induction of expression was not very strong. The described results demonstrate only the potential effect of NPs on innate immunity, and emphasize the need for further research in this direction.