*3.2. Cytotoxicity Studies of Cationic CNCs*

Assessment of toxicity in cell-based assays is a rapid, simple, and affordable approach to address the initial safety of nanomaterials with potential for biomedical application. These cytotoxic assays can be considered as a crucial part of the characterization of nanomaterials [35]. To evaluate the cytotoxicity of the cationic CNCs, we chose to perform MTT and NR assays using the firmly attached J774A.1 and BV2 cell lines because (i) these assays are largely used for screening of toxic and harmless compounds, (ii) quick analysis and low manipulation of attached cells after treatment and (iii) satisfactory reproducibility. In addition to cell lines, we tested the toxicity in a more relevant cell system, such as human cells PBMCs since the biological application of these nanomaterials can involve systemic circulation throughout the body. To assess the CNCs impact on cell viability in PBMCs, which remain loosely attached to the plate when not primed, we chose LIVE/DEAD® assay followed by flow cytometry analysis since it is an easy-to-use and sensitivity assay and PBMCs are not the most suitable cell type to access cytotoxicity by MTT or NR using a plate reader. Overall, our results indicated that CNCs have none or very low negative impact (<20%) on the cell viability assessed by all three cytotoxicity assays in all the cell types tested. However, results differed among CNCs and some cell types are more sensitive than others, depending on the assay used. Using MTT assay, which assesses cell viability by mainly mitochondria dehydrogenases, our data showed that in BV2 cells, the pristine CNC and CNC-AEM-1 did not decrease cell viability in any tested condition (Figure 3A). However, the METAC-modified CNCs did show a small decrease in cell viability, statistically significant, but not in a dose-response manner (Figure 3B). Using the same assay in J774A.1 cells, all the AEM-(Figure 3C) and METAC-modified CNCs (Figure 3D) did not impact negatively the cell viability in all tested concentrations. Interestingly, at the lowest concentration of CNC-METAC-1B (10 μg/mL), an increase in the conversion of tetrazolium to formazan was observed (Figure 3D). This effect does not indicate that there was an increase in cell viability above the control but rather an increase in the mitochondria dehydrogenase activity. This effect is not unknown since we have previously observed similar increases with other functionalized CNCs in other cell types, including MCF-7, a breast cancer cell line [23]. One reason for such an effect could be attributed to more tetrazolium dye entering into the cells, and therefore being more available to the dehydrogenases. A plausible explanation for potentially more dye entering the cells is that engineered nanomaterials, particularly high aspect ratio materials, can cause plasma membrane perturbation among other effects [36].

**Figure 3.** Cytotoxicity of unmodified and modified CNCs assessed by MTT assay. After 24 h of treatment, cell viability in BV2 and J774A.1 cell lines was determined spectrophotometrically. (**A**,**C**) pristine CNC and AEM modified and (**B**,**D**) METAC modified CNCs. Representative of 3 independent experiments, in triplicates. \* *p* < 0.01 compared to control, non-treated cells.

Data from NR assay indicated that in BV2 cells, CNC-AEM-2 appeared to cause an augmentation of NR dye uptake mainly in the highest concentrations (Figure 4A). A marginal negative effect on cell viability was observed in the cells treated with CNC-METAC-1B at the highest dose, 100 μg/mL (Figure 4B). Similar to what was observed with MTT assay, the J774A.1 cell line appeared to be less sensitive than BV2 cells. The AEM-modified CNC did not affect J774A.1 cells (Figure 4C) and only CNC-METAC-1B had a negative effect on the viability of these cells, although not a dose-response effect (Figure 4D). These differences could be attributed to potential physico-chemical changes on the nanomaterials since some nanomaterials might tend to form large agglomerates, sometimes larger than 100 nm size due to particle interaction with serum proteins [37], which was observed to have a significant effect on particle dispersion for certain materials. Furthermore, the cell lines used in this study were grown in a different medium, for instance, while J774A.1 cells were cultivated in RPMI, BV2 cells were grown in DMEM. Another aspect to consider is the differences in cellular responses was the biological differences among cell lines per se.

**Figure 4.** Cytotoxicity of unmodified and modified CNCs assessed by Neutral Red assay. After 24 h of treatment, cell viability in BV2 and J774A.1 cell lines was determined spectrophotometrically. (**A**,**C**) pristine CNC and AEM modified and (**B**,**D**) METAC modified CNCs. Representative of 3 independent experiments, in triplicates. \* *p* < 0.01 compared to control, non-treated cells.

We also evaluated the cytotoxicity of these modified CNCs using PBMCs and LIVE/DEAD® assay. Corroborating with what was observed with cell lines, the overall cell viability in primary cells PBMCs was not affected significantly and in a dose-response manner. Despite the variation of the data, the percentage of ethidium bromide-labeled cells did not change significantly in the presence of different concentrations of AEM- or METAC-modified CNCs, in comparison to the non-treated cells (Figures 5 and 6, respectively). As expected, H2O2 (positive control) increased the dead cell population as observed in the significant increases in the percentage of ethidium bromide labeling (Figure 5D,F). Although we observed that CNC-METAC-1B (Figure 6B) and CNC-METAC-2B (Figure 6D) displayed different flow cytometer spectrum from other CNCs, this difference appeared not to be related with an increase in cell death since it neither showed a characteristic peak in the H2O2 intensity range (Figure 5,D) nor a dose-response effect. We also did not observe differences in Calcein-AM staining between treatments and control. The SSC vs. FSC plot of non-stained cells was used for gating cells and excluding debris (Figure S6). A typical LIVE DEAD® flow cytometer SSC vs. FSC plot of staining cells (Ethidium bromide and Calcein-AM) treated of 100 μg/mL is displayed in the supplemental materials (Figure S7).

**Figure 5.** Flow cytometer plots from a representative experiment displaying the cytotoxic effects of unmodified (**A**) and AEM-modified CNCs (**B**,**C**) on PBMCs, as well as controls for gating (**D**). After 24 h of treatment, cell viability was determined by LIVE/DEAD® assay. The graph (**E**) displayed the average of fold of change in the (%) of dead cells (ethidium bromide staining) corrected by (%) dead cells in the control (non-treated cells), from at least 4 experiments. H2O2 500 μM, 4h was used as positive control for gating dead cells for the flow cytometer analysis (**D**,**F**).

**Figure 6.** Flow cytometer plots from a representative experiment displaying the cytotoxic effects of METAC-modified CNCs (**A**–**D**) on PBMCs. After 24 h of treatment, cell viability was determined by LIVE/DEAD® assay. The graph (**E**) displayed the average of fold of change in the (%) of dead cells (ethidium bromide staining) corrected by (%) dead cells in the control (non-treated cells), from at least 4 experiments.
