Table 1.

Studies focusing on local and systemic toxicity of pure 2D materials.

Type of material	Treatment type	Cell type/Animal model	Effect	Reference
Graphene nanoplatelets (GNPs)	In vitro	Human bronchial epithelial cell line (BEAS-2B)	Dose-dependently reduced cell viability, which was accompanied by an increase in the portion of cells in the subG1 and S phases, downregulated the generation of ROS, suppressed ATP production, damaged mitochondria, and elevated levels of autophagy-related proteins	[181]
	In vivo	Mice	Subchronic inflammatory response
Graphene quantum dots (GQDs)	In vitro	Cancer cell lines (KB, MDA-MB231, A549) Normal cell line (MDCK)	No acute toxicity or morphological changes	[182]
Graphene quantum dots (GQDs)	In vivo	Mice	GQDs mainly accumulated in liver, spleen, lung, kidney, and tumor sites after intravenous injection.
			No toxicity to the treated animals or organ damage/lesions to treated mice after 21 days of administration at 5 mg/kg or 10 mg/kg dosages
	In vitro	HeLa (A549) cells	Low cytotoxicity	[212]
	In vivo	Mice	No material accumulation in main organs and fast clearance of GQDs through kidney
GO and rGO	In vitro	HEK 293T cells	Small and large sizes of G and GO significantly reduced the cell viability and increased DNA damage, accompanying with activated reactive oxygen species (ROS) generation and induced various expressions of associated critical genetic markers	[183]
	In vivo	Zebrafish	G showed stronger ability to decrease the survival rate and induce the acute toxicity, while GO showed obvious toxicity in terms of DNA damages, ROS generation, and abnormal gene expressions
	In vitro	Luminous Tox2 & RecA bacterial strains	G and GO caused strong acute toxicity on Tox2 bacteria; G was more toxic than GO and showed size dependent effect with toxicity increasing with increase in size. GO induced mild genetic toxicity on RecA bacteria
	In vitro	Murine macrophage RAW 264.7 cells	ROS-mediated toxic effects with pristine graphene	[184]
	In vitro	E. coli; S. aureus	Bacteria cell membrane damage: GO at a concentration of 85 μg/ml could suppress the growth of E. coli after 2 h incubation at 37 °C	[185]
	In vivo	Mice	Chronic toxicity with high dose (0.4 mg) in lung, liver, spleen, kidney	[189]
	In vitro	E. coli, Pseudomonas aeruginosa	No noticeable antimicrobial effect with GO alone	[187]
	In vivo	Mice	Accumulates in reticuloendothelial system (RES) organs, such as liver and spleen at early time points after IV injection	[213]
	In vitro	Human platelets	Evokes aggregatory response, which may play a role in hemostasis and thrombus formation	[180]
	In vivo	Mice	Thrombogenic
	In vitro	HeLa cells	Dose-dependent toxicity	[184]
	In vivo	Zebrafish	Polyacrylic acid (PAA) was used to functionalize GO to reduce its toxicity to cells
	In vitro	A549 human epithelial cells	GO does not enter A549 cell and has no obvious cytotoxicity, but can cause a dose-dependent oxidative stress in cell and induce a slight loss of cell viability at high concentration	[214]
	In vivo	Mice	Induce the mitochondrial generation of ROS, activate inflammatory and apoptotic pathways, and result in severe and persistent lung injury	[184]
	In vitro	U87 and U118 glioma cell lines	Both types of platelets reduced cell viability and proliferation with increasing doses, but rGO was more toxic than GO with rGO increasing the level of apoptotic markers in treated tumors	[215]
	In vivo	Mice	Short-term repeated GO exposure can cause obvious intraocular inflammation, an incrassated corneal stromal layer, cell apoptosis in the cornea, iris neovascularization and significant cytotoxicity of rat corneal epithelial cells (rCECs), while RGO causes no significant ocular toxicity in mice	[190]
Laponite® XLG, nanohybrids	In vitro	Cancer cells (KB human epithelial carcinoma cells, MCF-breast cancer cells, CAL-72 osteosarcoma cell lines)	Cytotoxicity towards cancer cells	[216]
	In vivo	Mice	Absence of organ toxicity 24 h and half a month post IV injection
	In vitro	Human mesenchymal stem cells (hMSCs)	Non-toxic at concentrations less than 1 mg/ml	[192]
	In vivo	Rats	Implantation of Laponite bioceramics does not show acute systemic toxicity or irritation to normal skin of rats	[193]
MMT	In vitro	INT-407 (intestinal) cells	MMT could cause cytotoxic effects in a concentration- and time-dependent manner and at high concentration after long-time exposure	[195]
	In vivo	Mice	MMT absorbed within 2 h but did not accumulate in any specific organ or showed any remarkable toxicity
	In vitro	HUVE, N1E-115 and ROC-1 cell lines	Decrease cell viability in HUVECs exposed for 24 h to 0.1 mg/ml but limited results in neuronal cell lines N1E-115 and ROC-1	[217]
	In vitro	CHO cells	Reduction in cell viability after being exposed to 1 mg/ml for up to 24 h
MMT nanocomposite (MMN)	In vivo	Broiler chicks	Addition of 3 g MMN/kg AF-contaminated diet diminished the adverse effects of AF on most relative organ weights, hematological values, serum and liver biochemical values and enzymatic activities associated with aflatoxicosis in broiler chicks	[218]
BP nanosheets	In vitro	Human bronchial epithelial cells	BP nanosheets decrease viability of these cells in a time- and dose-dependent manner via interference with mitochondrial membrane potential, leading to an increase in intracellular ROS, activation of caspase-3, and apoptosis	[219]
	In vivo	Mice	Single injection of BP nanosheets does not cause toxicity in a short period of time, whereas multiple injections of BP nanosheets exert adverse effects on liver and renal function
BP quantum dots (BPQDs)	In vitro	HeLa cells	At high concentration (200 μg/ml) exhibit significant apoptotic effects	[211]
	In vivo	Mice	BPQD can transiently induce oxidative stress, including lipid peroxidation, reduction of catalase activity, DNA breaks, and bone marrow nucleated cells (BMNC) damage, but recovered gradually to healthy levels and no pathological damage to organs is apparent
Black phosphorus (BP)-quantum dots (QDs)	In vitro	Kidney organoid	Moderate toxicity	[209]
	In vitro	Huma renal tubular epithelial cells	Nephrotoxicity
	In vivo	Mice	Nephrotoxicity - found to cause insulin insensitivity and endoplasmic reticulum strees in the kidney
MoS2, WS2, and WSe2	In vitro	Lung epithelial carcinoma cells	WS2 elicit slightly more toxic responses whereas MoS2 and WS2 are safe and less hazardous	[199]
MoS2 nanosheets	In vitro	NIH-3T3 murine embryo fibroblast cells and the human adipose derived mesenchymal stem cells	Mild toxicity	[220]
		Human embryonic lung fibroblast (HELF) cells	Bovine serum albumin (BSA) coating diminished the toxic effect, and both coated and uncoated NPs promoted the proliferation of HELF cells, which, as a pathological process, can lead to idiopathic pulmonary fibrosis
	In vitro	HepG2 cells	Cell membrane disruption: ROS level and the mitochondrial membrane potential were altered in the treated cells, suggesting oxidative stress and apoptosis induction; inhibition of ABC transporter
	In vitro	BEAS-2B immortalized human bronchial epithelial cells; THP-1 human monocytic cells	Cell viability unaltered with proinflammatory effect for aggregates and low effect for nanoforms
	In vivo	Mice	Proinflammatory effect for aggregates and low effect for nanoforms
MoS2 nanofilm and microparticles	In vitro	PANC1 human pancreatic cancer cells; 293 human embryonic kidney cells; human pancreatic duct epithelial (HPDE) cells; human mammary epithelial (HMLE) cells; SUM159 mesenchymal triple-negative breast cancer cells; MDAMB-231 invasive ductal breast carcinoma cells	Cell viability unaltered up to 0.016 mg/ml, slight decrease in viability for higher concentrations
	In vivo	Guinea pigs	No skin allergy symptoms
Molybdenum trioxide (MoO3) nanoparticles	In vitro	Human MCF-7 (breast cancer) and HepG2 (hepatoma)	Showed a dose-dependent decrease in viability at concentrations (25–0.625 μg/ml)
		C18–4 spermatogonial stem cell line	Cell membrane integrity disruption
		BRL 3A cells (immortalized rat liver cells)	Mildly toxic effect at 250 μg/ml but significant LDH leakage present at the concentration of ≥100 μg/ml.
MoO2 and MoO3 nanocolloids	In vitro	NIH/3T3 cell line	Decreased viability in a concentration dependent manner associated with a change in the redox status of the cell, resulting in oxidative stress induction
MoO3 nanoplates	In vitro	Human breast cancer cells (MCF-7 and the invasive type MCF-7 with the CD44high/CD24low phenotype) and human keratinocyte (HaCaT)	HaCaT cells did not show decreased viability, but both MCF-7 lines were susceptible and likely to involve the loss of the mitochondrial membrane potential and an induction of oxidative stress
hBN nanosheets	In vitro	E. coli DH5α	Degradation of bacterial cell membranes (inner and outer layers)	[201]
hBN nanoparticles	In vivo	Rats	Damage in the liver, kidney, heart, spleen, and pancreas at 1600 and 3200 μg/kg doses	[205]
hBN nanosheets and nanoparticles	In vitro	Osteoblast-like cells (SaOS2)	Decrease in cell viability in presence of both nanosheets and nanoparticles with the smallest size likely attributed to internalization and activation of ROS	[205]
hBN flakes	In vitro	Mouse hippocampal cell line (mHippo E14)	No cytotoxic effects at hBN concentration lower than 22 μg/ml and favored the cell survival after exposure to doxorubicin
Ti3C2 and Nb2C Mxene quantum dots	In vitro	Human umbilical vein endothelia cells (HUVECs)	Ti3C2 QDs could induce cytotoxicity to HUVECs at 100 μg/ml	[221]
MXene flake/Cu nanocomposite	In vitro	3T3 mouse fibroblasts and HEK293 cells	No toxic effects	[222]
Ti3C2Tx MXene film	In vitro	Mouse pre-osteoblast cell line MC3T3E1	No cytotoxicity	[207]
	In vivo	Rat calvaria	No toxic or inflammatory effects; improves osteogenesis
	In vivo	Mice	No adverse effects or pathological toxicity (20 mg/kg)
MXene, Nb2C-PVP nanosheets				[222, 223]
MXene nanosheets	In vivo	Avian embryos	Adverse effect on the early stage of embryogenesis as ~46% of MXene-exposed embryos died during 1–5 days after exposure and may inhibit angiogenesis of the chorioallantoic membrane of the embryo after 5 days of incubation; seven genes that are key regulators of cell proliferation, survival, cell death and angiogenesis were deregulated in brain, heart, and liver tissues	[224]
	In vivo	Zebra fish embryos	LC50 of Ti3C2Tx was greater than 100 μg/ml, it can be classified as within the “practically nontoxic” group according to the Acute Toxicity Rating Scale by the Fish and Wildlife Service (FWS)	[225]