## 3 Results and Discussion
### Results
#### 3.1.2 Evaluation of Toxicity of CQDs and SQDs
According to our spectrophotometry data, no changes in nanodot content were observed after short-term (1 to 6 h) exposure of 3T3-L1 cells to CQDs at doses between 0.2-25 mg\(\cdot\)L\({}^{-1}\), suggesting delayed incorporation of nanodots in the cells in the presence of serum (Figure 6 and Figure S5). Aberrant SQD accumulation in death cells cultured in FBS prevented us from measuring fluorescent content in 3T3-L1 cells exposed to these NPs. Toxicity of CQDs and SQDs in HepG2 Cells  
We extended our studies on CQDs and SQDs to HepG2 cells, which are considered a suitable in vitro model for toxicological studies [27]. In fact, a variety of NPs have been designed to target these cells [28]. * Defining the optimal cellular conditions prior to cytotoxicity assay. Preliminary studies using this cell line revealed that HepG2 cells required the presence of serum to maintain proper cell viability (Figure S6). To be more specific, FBS concentrations below 5% reduced, by more than 50%, cell viability (Figure S6). Thus, experiments aimed at assessing the effects of nanodots on HepG2 cells were carried out in media containing 10% FBS. * MTT cell viability assay. These cytotoxicity studies showed that high CQDs concentrations compromised cell viability (Figure 7A). This was particularly noticeable in the cell cultures exposed to 1600 mg\(\cdot\)L\({}^{-1}\) for 24 h, wherein cell viability was reduced by up to 22.1% (Figure 7A). In contrast, exposure to SQDs for 24 h was toxic to HepG2 cells at concentrations as low as 0.2 mg\(\cdot\)L\({}^{-1}\), yet cell viability remained above 84% for all the doses tested (Figure 7B). Figure 6: Fluorescence emission in 3T3-L1 cell cultures exposed for 6 h to increasing concentration of CQDs in the absence of serum. Control cells were cultured in medium (DMEM) alone. Measurements were carried out at fluorescence excitation wavelength of 365 nm and emission wavelength of 450 nm. Data (mean \(\pm\) SEM of at least three independent experiments with three replicate wells per treatment) were analyzed by one-way ANOVA multiple comparison test followed by [PERSON]’s LSD test. Extracellular-Quantum Dot Interaction Studies. Influence of the Culture Media on Nanodot Behavior  
Next, we explored the possible interaction between different microenvironments and nanodots by monitoring the fluorescence signal of CQDs and SQDs in the presence of either DMEM (specific for 3T3-L1 cells), MEM (specific for HepG2 cells), or FBS (for both cell types). As shown in Figure 8, CQD emission intensity increased when these nanodots were diluted in DMEM, MEM, or FBS, indicating the occurrence of an electron transfer reaction. Regarding SQDs, quenching of fluorescence was observed when these nanodots were diluted in DMEM or FBS, but not MEM (Figure 8). Figure 7: Cell viability data in carcinoma human hepatocellular cells (HepG2) cells cultured in the presence of 10% FBS and exposed to CQDs (**A**) and SQDs (**B**) at concentrations ranging from 0–1600 mg:L\({}^{-1}\) for 24 h. Control cells were cultured in medium (DMEM) alone. Cell viability was measured by the MTT assay. Data (mean \(\pm\) SEM of at least three independent experiments with three replicate wells per treatment) were analyzed by one-way ANOVA multiple comparison test followed by [PERSON]’s LSD test. Significant differences are indicated as * \(p<0.05\), ** \(p<0.01\), *** \(p<0.001\) vs. control; # \(p<0.05\), *** \(p<0.001\) vs. 400 mg:L\({}^{-1}\); $$$$ \(p<0.001\) vs. 800 mg:L\({}^{-1}\). We also tested the behavior of CQDs in response to the combination of DMEM and FBS, as that employed for 3T3-L1 cell growth in culture. As shown in Figure S7, a shoulder in the characteristic emission band of CQDs was observed, though this shoulder disappeared after continuous irradiation for almost 7 min. This approach defines the best conditions to compare toxicity effects of NPs on cell types which are also dependent on the extracellular media, as shown in Figure 2:0