The extensive potentials of CNM for biomedical application have been highlighted, including antibacterial [51,52], cell adhesion and proliferation [53,54], inducing osteogenic [55,56], osteoconduction [57,58], and osseointegration effects [59]. However, biocompatibility, which often shows contradictory or inconclusive results, has issues that should be elucidated. The biocompatibility of CNMs often time-, size-, and dose-dependently works, however, it varies by raw materials, fabrication methods, and physicochemical functionalization [60,61,62]. Since it is difficult to draw accurate conclusions, we intend to provide guidelines for later studies by comparing relevant studies.

GO’s dose-dependent cytotoxicity on bone marrow-derived stem cells (BMSCs) and assessed toxicity mechanisms were investigated [63]. GO significantly inhibited cell viability at ≥2.5 µg/mL by interrupting membrane integrity. At the same concentration, cell apoptosis was one-and-a-half-fold increased but did not hinder the cell proliferation cycle significantly. Furthermore, ≥2.5 µg/mL of GO induced intracellular ROS generation, inducing ROS-associated damage, and caused cell dysfunction which was assessed by mitochondria membrane potential (MMP) loss. Western blotting showed upregulation of Cleaved Caspase-3, LC3-II/I, and Beclin-1 and downregulation of Bcl-2 and Caspase-3, indicating that GO-mediated cytotoxicity is related to mitochondrial autophagy and triggering cellular apoptosis. The hemolytic and cytotoxic effects of GO, which are synthesized in various methods, showed varying results according to their sizes, particulate states, surface charges, and oxygen contents [64]. Hemolysis and morphologies of red blood cells (RBCs), intracellular ROS generation, and fibroblast viability were significantly different according to the fabrication methods, suggesting that the particulate state of G materials has a profound impact on cytotoxicity. The cytotoxicity and genotoxicity of different CNMs are proven to be material-specific and cell-specific with a general trend for biocompatibility (ND > carbon powder > MWCNT > SWCNT > fullerene) [50]. For example, macrophages are more cytotoxic than neuroblastoma cells, and CNT and MWCNT tend to cause DNA damage in mouse embryonic stem cells by ROS generation [50]. NDs possess minimal cytotoxicity because their chemical inertness does not release any toxic chemicals and round morphologies [65]. Carboxylated NDs were shown to not exhibit cytotoxicity or genotoxicity on human cell lines including liver, kidney, intestine, and lung cell lines, which are major accumulation organs after the nanoparticles are injected [66]. On the other hand, fullerene shows significant cytotoxicity mainly contributing to ROS generation. Fullerenes under ambient water conditions can generate superoxide anions that are responsible for membrane damage and subsequent cell death [67].

For clinical usages including drug delivery, bioimaging, biosensing, and other theragnostic applications, in vivo toxicity of CNMs has been intensively studied. To understand the potential threat of CNMs in the body, biodistribution and accumulation mechanisms should be elucidated. The accumulation of GO in mouse lung induced oxidative stress by an increase of mitochondrial respiration and activated inflammatory and apoptotic pathways [68]. On the other hand, surface functionalization and chemical modification have been introduced to enhance the biocompatibility and biofunctionality of G materials [69,70,71]. The PEGlyated GO and rGO were developed for oral and intraperitoneal (i.p.) injection, and the biodistribution was investigated [72]. After seven days, oral administration could not be adsorbed by organs and rapidly excreted, however, i.p.-administered PEGlyated GO and rGO were accumulated most highly in the liver and spleen but were finally engulfed by phagocytes in size- and surface coating-related manner. The results indicated that no significant toxicity was found in serum biochemistry, complete blood panel test, and histological analysis, indicating that PEGylation can facilitate biocompatibility of G materials. In a similar study, intravenous (i.v.) injected G quantum dots (GQDs) did not exhibit to vital organs of rats, although slight reduction of platelets and monocyte and eosinophil fractions occurred, which were soon normalized [73]. After respiratory administration, CNT remains in the lungs for months or even years and is eliminated through the gastrointestinal (GI) tract. It does not cross the pulmonary barrier or get absorbed in the GI tract [74,75]. A single intratracheal instillation of SWCNT triggered epithelial granulomas and interstitial inflammation, developing peribronchial inflammation and necrosis [76].