## 3 In Vitro and In Vivo Toxicity of CDs
They injected CDs into mice through three different administration routes (2.5 mg CDs/kg), i.e., intravenous (IV), intramuscular (IM), and subcutaneous (SC) administrations, and measured biodistribution and urine clearance of CDs [117]. They observed that the injected CDs could be effectively and rapidly excreted through urine for all three administration routes owing to ultrasmall particle sizes (d = \(\sim\)3 nm) with the clearance rate of IV \(>\) IM \(>\) SC (Figure 7a). In addition, their biodistribution study demonstrated that the majority of CDs were accumulated at the kidneys at 1 h after administration in the order IM \(>\) IV \(>\) SC (Figure 7b), and the remaining CDs in the mice body were very little in all organs at 24 h after administration for all administration routes, indicating that most of the CDs were excreted as urine from the body (Figure 7c). [PERSON] et al. prepared CDs using a simple solvothermal approach and citric acid as the carbon source. They conducted a comprehensive histopathological assay to evaluate the safety and biocompatibility of CDs after sacrificing mice on the first and seventh day after IV administration (administration dose = 25 mg CDs/kg) (Figure 7d) [113]. The analysis involved hematoxylin and eosin (H&E)-stained tissue slices from major organs and revealed no signs of inflammation, necrosis, or other pathological changes up to the seventh day after administration; the normal tissue structures were maintained with well-organized cardiac myocytes, clearly defined liver lobules, intact lung structure, preserved renal glomeruli, and healthy splenic corpuscles. [PERSON] et al. synthesized CDs using citric acid and urea as the carbon precursors in a solvothermal method and also examined histopathological assay using H&E-stained sections of the kidneys, heart, liver, spleen, and lung, and observed no significant signs of organ damage or inflammatory lesions compared to the control groups with no CDs [118]. [PERSON] et al. synthesized CDs through a solvothermal reaction of citric acid and PEI as the carbon precursors and then conjugated chlorin e6 (Ce6) to obtain Ce6-modified CDs. They performed the histopathological assay of CDs using H&E-stained sections of excised major mouse organs (kidneys, heart, liver, spleen, and lungs) and observed no significant inflammation and damage in the mice's organs compared with those of the control groups, which received phosphate-buffered saline (PBS) [39]. All these in vivo studies demonstrated that CDs are safe and nontoxic. However, almost all toxicity studies on CDs performed so far are acute and single-dose experiments. Therefore, chronic toxicity studies are essential to reveal the potential long-term toxicity of CDs because multiple dosages of CDs are generally required for disease treatments. The CDs exhibited low or negligible toxicity up to 90 days after IV administration (single dose = 20 mg/kg mice) [119]. Mice that received daily doses of 100 mg/kg over 7 days also exhibited low toxicity up to 90 days [120], indicating the low toxicity of CDs. The CDs could adjust the immune function of BALB/c mice by inducing immune Th1 and Tc cell responses, but these effects were not enough to induce the morphological change in immune organs [121], indicating the safety of CDs for the immunological system of mice. However, the mechanism by which CDs modulate the immune system remains unclear. Therefore, to facilitate the commercialization of CDs for clinical applications, more studies are needed, and it will be valuable to develop standard toxicity evaluation protocols for acute and chronic (>90 days) toxicity and immunological assays.