The field of nanomedicine has attracted attention in recent years due to the advantages it offers in various biomedical applications. Along with this attraction, the need to investigate interactions and effects of nanomaterials on biological systems has been increasing. Several carbon-based nanoparticles have been in the focus of research and among these particles, ND stand out as the most biocompatible material [7–11]. We aimed to assess the effects of ND on macrophages using the macrophages cell line, J774A.1 and primary BMDM.
Our results showed some variations in the effects of ND between the two cell types. Cell lines have been extensively used for cytotoxicity studies. However, the continuous culturing of cells in vitro results in loss of genes that might not be required for cells’ survival and therefor, may not represent the response of normal cells in vivo [31, 46]. Previous studies have reported significant differences between cell lines and primary cells . Our primary cells, BMDM were used in this study to confirm and compare results from the macrophage cell line and to observe the effects of ND on these cells during their differentiation.
The microscopic images showed that these cells internalized ND into their cytoplasm without any significant effects of the cell shape. However, ND-treated cells appeared larger in these images due to their darker color as compared to the transparent, control cells. The FACS results showed that these cells decreased in size in response to ND (26–32% reduction in size). Other studies showed that macrophages and other cell types increased in size in response to ND treatment [48, 49]. In these studies, the size of the ND was 10 times smaller than our ND (< 10 nm), which tend to cause more harmful effects on cells [17, 50]. The FACS results also showed an increase in cellular granularity which has also been reported previously . The internalized ND remained in the cytoplasm of these cells which has also been reported in previous studies but for other cell types [12, 13].
The bright fluorescence of ND was efficiently captured through the fluorescence microscope and FACS. The fluorescence intensity increased with increasing the concentration of ND or increasing the treatment intervals. The intrinsic fluorescence of ND arises from the presence of the negatively charged nitrogen vacancy centers . This stable fluorescence makes ND suitable for imaging and tracking applications . The uptake of these particles was shown to be dependent on the concentration and treatment intervals in addition to the size of the particles as well as the cell types [13, 53]. The uptake mechanism of nanoparticles depends on their specific characteristics and on the cell type . We used different endocytic pathway inhibitors to study the uptake mechanism of ND in these cells and our results showed that the uptake was reduced by about 40% when using the inhibitor of actin-dependent pathways and by about 20% when using the inhibitor of clathrin-dependent pathways. Other cell types have been reported to internalize ND through clathrin-dependent pathway . Professional phagocytes such as macrophages can endocytose varying sizes of particles through phagocytosis, which is actin-dependent, but this does not preclude endocytosis through the other pathways. Huang and coworkers showed that ND internalization in RAW264.7 macrophages was reduced by 23% using the same clathrin pathway inhibitor but at lower concentrations . However, they did not report any effect of the actin pathways inhibitor.
The fact that these particles can be clearly detected inside cells is beneficial specially for tracking and imaging applications. However, this feature can be problematic because they can interfere with colorimetric and fluorometric assays. We showed that ND can absorb light at different wavelengths, and they also interfere with fluorescence signals at different wavelengths and therefor one should pay attention when assessing the effects of these particles on cells for example for viability assays. Interference of nanoparticles with toxicity assays has previously been reported for different particles [56, 57]. We used the MTS assay because the detection of cells’ metabolic activity does not require lysing of cells to release the produced formazan. Our results showed no significant reduction in cell viability after exposure to ND with different concentrations and for different time points. Similarly, previous studies showed no significant effects of ND on different cell types [7, 55]. These studies used the MTT assay which require measurement of the absorbance at 570 and 690 nm, which we have seen to have ND interference. A more recent study showed a reduction of about 14% in cell viability in response to ND-COOH, which were functionalized from smaller (4–5 nm) pristine ND .
Expression of inflammatory cytokines and chemokines were studied at the mRNA and the protein level, and the results showed no significant difference between ND-treated and untreated cells. Although we did not see statistically significant changes in genes expression, the results clearly showed differences in the responses between the two cell types. The proinflammatory cytokines and chemokines, interleukin (IL)1β, CCL2, and CXCL2 were significantly upregulated in 3 out of the 4 replicated experiments using the J774A.1 cells. However, only CXCL2 was upregulated in BMDM. Previous studies have shown different results regarding the regulation of the expression of various pro- and anti-inflammatory cytokines. Huang et al. showed that small (2–8 nm) ND had no effects on the expression of IL6, TNFα, and iNOS in a murine macrophage cell line . Another study also showed downregulation in the expression of IL1β, TNFα, CXCL2, CCL2, PDGF, and VEGF for the same cell type (RAW 264.7) in response to different sizes of ND . Huang and coworkers also reported no significant production of pro-inflammatory cytokines from these cells . Expression of TNFα but not IFNγ increased in response of human monocytes to ND without upregulating the expression of activation markers . On the other hand, carboxylated ND have been shown to induce inflammatory cytokines in monocytes . However, this study used a different cell type and much smaller particles as compared to the ND we used. Another recent study also showed significant expression of inflammatory cytokines from a monocyte cell line . The variations in the change in gene expression can be explained by the type of cells and the type of particles being studied
The ability of different nanoparticles to modulate or inhibit the immune functions of macrophages has been reported and therefore, we aimed to study the effects of ND on two main macrophage functions. Cells were pre-treated with ND before exposing them to conventional macrophage activator, LPS. The response of the two cell types was then assessed using RT-qPCR; the results showed that both cell types, ND-treated or untreated, were able to respond normally to LPS. To our knowledge, the effect of ND on macrophage ability to respond to LPS has not been investigated. The ability of RAW 264.7 cells has been shown to be attenuated in response to CpG (TLR9 ligand) after treatment with gold nanoparticles . Further studies are still needed to investigate the effects of ND on the response of macrophages to other immunogens and at other ND concentrations.
The endocytic activity of macrophages after treatment with ND has also been studied and the results showed a significant decrease the ability of these cells to internalize dextran particles. The endocytic activity of macrophages after exposure to ND has not been studied but other nanoparticles have been shown to reduce this activity in macrophages [63–65]. In these studies, the nanoparticles did not induce significant reduction in cell death or inflammatory response, rather they showed interference in macrophages functions such as response to LPS and phagocytic activity. These results show that different nanoparticles might be used to manipulate the functions or responses of immune cells for different therapeutic purposes.
The primary macrophages we used in this study were prepared from mice bone marrow under M-CSF. We studied the effects of ND on the differentiating cells and the results showed that cells’ morphology did not change in response to ND treatment. In addition, the percent of cells expressing macrophages surface markers (CD11b and F480) did not change. However, the number of surface markers expressed on these cells were reduced significantly which may indicate an effect on macrophage function. These markers are required for various macrophage functions including cell adhesion, spreading, and migration . Previous studies showed significant changes in the ability of macrophages to phagocytose opsonized and non-opsonized beads after exposure to different nanoparticles . An in vivo study in chicken embryos showed that ND may interfere with cells differentiation through downregulation of growth factors .
Several previous studies investigated the effects on different types of ND on cell viability while fewer reports investigated their effects of macrophages function. To our knowledge, the effects of ND on BMDM during and after differentiation have not been investigated. Further studies are needed to investigate the effects of ND on macrophage functions and the mechanisms by which these particles are interfering with macrophages functions. The response of different cell types may vary significantly and therefore it is essential to study interactions of different nanoparticles in different systems before concluding their toxic/nontoxic effects. In addition, it is also important to note the ability of these particles to interfere with both the absorbance and fluorescence signals which may lead to false results in toxicity studies.