## 3 In Vitro and In Vivo Toxicity of CDs  
CDs are mainly composed of nontoxic C with additional H, O, and N, and their exact compositions depend on carbon precursors. CDs are free from toxic heavy metal ions and thus, promising nontoxic nanoprobes, making them potential candidates for biomedical applications. Ideally, upon administration of CDs for in vivo applications, they should perform their intended functions effectively and subsequently be excreted from the body without causing any adverse effects. Both in vitro and in vivo toxicities of CDs, which are dependent on their chemical composition, size, surface charge, and functionalization, have been widely studied [104; 105; 106]. As schematically illustrated in Figure 5, for instance, the positively charged CDs (with surface functional groups such as -NH\({}_{2}\)) tend to have higher cytotoxicity compared to negatively charged CDs (with surface functional groups such as -OH and -COOH) or neutral charged CDs (with surface functional groups such as -H) [107]. This can be attributed to a greater capacity of positively charged CDs to interact with negatively charged phospholipids of cell surfaces and their subsequent higher cellular uptake [108]. Furthermore, surface functionalization with biocompatible molecules such as polyethylene glycol (PEG) can significantly enhance their stability and reduce in vivo toxicity [109]. Figure 5: Schematic representation of toxicity related surface conditions of CDs. The in vitro toxicity is characterized by measuring the cell viability. The common methods include the methylthalazole tetrazolium (MTT), cell counting kit (CCK)-8, and water-soluble tetrazolium (WST)-1 methods [110; 111; 112]. Until now, numerous studies have demonstrated that CDs have excellent cell viability in vitro. For example, [PERSON] et al. prepared CDs using citric acid, urea, and formamide as carbon sources in a solvothermal method and analyzed the cytotoxicity of CDs using human lung cancer (A549) and Henri-etta Lacks (HeLa) cancer cells [113]. Their study revealed that the cell viability remained above 85% even at high CD concentrations (400 \(\upmu\)g CDs/mL) after both 12 and 24 h of incubation, demonstrating the low cytotoxicity of CDs (Figure 6a,b). [PERSON] et al. prepared CDs using citric acid and formamide as carbon sources in a solvothermal method and examined the in vitro cytotoxicity of CDs at different CD concentrations (0, 200, 400, 600, and 800 \(\upmu\)g CDs/mL) using National Institutes of Health (NIH) 3-day transfer (3T3) cells after 24, 36, and 72 h incubations with CDs (Figure 6c) [114]. Their results demonstrated that the cell viability remained above 90% for all CD concentrations. [PERSON] et al. prepared CDs using gallic acid, carbamide, and PEG400 as carbon sources in a microwave method and demonstrated that CDs exhibited negligible cytotoxicity towards hepatoblastoma (HepG-2), Michigan Cancer Foundation (MCF)-7, and mouse fibroblast (L-929) cell lines at concentrations ranging from 2 to 100 \(\upmu\)g CDs/mL over 24 h incubation with CDs (Figure 6d) [115]. Their results exhibited that the cell viability remained approximately 95% for all cells, demonstrating very low cytotoxicity of CDs in vitro. The in vivo toxicity of CDs using living organisms includes various assays, including biodistribution, clearance, and major organ toxicity (liver, heart, kidney, lung, and  
Figure 6: In vitro cell viability of (**a**) A549 and (**b**) HeLa cells after incubation with different concentrations of CDs for 12 and 24 h [113]. (**c**) Cell viability of NIH 3T3 cells after incubation with different concentrations of CDs for 24, 36 and 48 h [114]. (**d**) Cellular cytotoxicity of HepG-2 cells, MCF-7 cells, and L-929 cells after incubation with Green-CDs [115]. spleen) [116]. The biodistribution and clearance of CDs from the body depend on the administration route. [PERSON] et al. synthesized CDs via laser ablation of a carbon target in the presence of water vapor and argon, followed by surface modification with PEG (MW = \(\sim\)1500 amu).