## 3 Results and Discussion
### Results
#### 3.1.2 Evaluation of Toxicity of CQDs and SQDs
As shown in Figure 4, 3T3-L1 cells exhibited significantly higher levels of total intracellular fluorescence than control cells (i.e., exposed to medium alone) when incubated in the presence of CQD doses above 10 mg-L\({}^{-1}\) for 6 h. Lower concentrations of CQDs (0.2 mg-L\({}^{-1}\)) appeared too low to detect increases in intracellular fluorescence levels (Figure 4). Notably, nanoparticle incorporation into the cells did not only depend on the concentration, but also on the time of exposure to the nanodots. Thus, at shorter incubation times (1 h), only higher CQD doses (25 mg-L\({}^{-1}\)) evoked significant increases in cell fluorescence (Figure S4). Regarding SQDs, accumulation of NPs could be observed by confocal microscopy in the cell debris remaining in the cultures after treatment (Figure S3), which rendered aberrant measurements of intracellular fluorescence levels by spectrophotometry (data not shown). Figure 4: 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. Significant differences are indicated as \({}^{*}p<0.05\), \({}^{***}p<0.001\) vs. control. Figure 3: Confocal microscopy images obtained from 3T3-L1 cells exposed to 25 mg-L\({}^{-1}\) of CQDs or SQDs in the absence of serum for 24 h. Control cells were cultured in medium (DMEM) alone. Nuclei (blue) were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). _Condition 2_: Presence of serum in cell culture:  
* MTT cell viability assay. The response of 3T3-L1 cells to NPs was also explored at long-term exposure times. We observed that, when administered for 24 h to 3T3-L1 cells in the presence of serum, CQDs at intermediate doses (25 mg\(\cdot\)L\({}^{-1}\)) slightly, yet significantly, decreased cell viability (Figure 5A). Nevertheless, even at concentrations of CQDs as high as 1600 mg\(\cdot\)L\({}^{-1}\), 3T3-L1 cell viability remained above 85% (Figure 5A). In clear contrast, the presence of serum in the culture media seemed to increase SQD-induced toxicity, compared to that observed in FBS-free culture media. Thus, all the SQD doses examined diminished cell viability, and the reductions in this parameter were higher in the presence (Figure 5B) than in the absence (Figure 2B) of FBS. Specifically, when administered in the absence of FBS, SQDs at 100 mg\(\cdot\)L\({}^{-1}\) reduced, by 19.4%, cell viability (Figure 2B), compared to the 66.5% reduction observed for this SQD concentration in FBS-exposed cell cultures (Figure 5B). Figure 5: Cell viability data in 3T3-L1 cells cultured in the presence of 10% FBS and exposed to CQDs (**A**) and SQDs (**B**) at concentrations ranging from 0–1600 mg\(\cdot\)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 \"\" \(p\) \(<\) 0.01, \"\" \(p\) \(<\) 0.001 vs. control; # \(p\) \(<\) 0.05, \"\" \(p\) \(<\) 0.01, and \"\" \(p\) \(<\) 0.001 vs. 0.2, 25, or 400 mg\(\cdot\)L\({}^{-1}\), respectively; $$5 \(p\) \(<\) 0.001 vs. 0.2 mg\(\cdot\)L\({}^{-1}\). Cell uptake assay. The presence of serum in the culture media also altered the time course of nanodot accumulation in 3T3-L1 cells.