Nanoscience has matured significantly during the last decade as it has transitioned from bench top science to applied technology 
. Presently, silica nanoparticles are widely used in biomedical applications as promising carriers for drug delivery or gene therapy. Thus, the endothelial cells could be primarily exposed to silica nanoparticles by intravenous administration. However, biological or cellular responses to silica nanoparticles are still poorly understood. DNA damage response (DDR) involved the sensing of DNA damage followed by transduction of the damage signal plays an important role in the network of cellular pathways. To our best knowledge, the possibility mechanisms of DDR pathways triggered by silica nanoparticles caused the toxic effect of endothelial cells has not been investigated. Our findings demonstrated that direct exposure of HUVECs to silica nanoparticles induced DDR leading to activate the Chk1-dependent G2/M checkpoint signaling pathway, resulted in a series of endothelial cells toxic effect.
Currently, cell uptake of nanoparticles is an important issue in designing suitable cell-tracking and drug-carrier nanomaterials systems 
. LSCM and TEM results showed that silica nanoparticles were internalized into endothelial cells after 24 h exposure (). In addition, our previous study confirmed that the silica nanoparticles could internalized into the cells and dispersed in cytoplasm and deposited inside mitochondria 
. To gain closer mechanistic insight into silica nanparticles-induced biological effects, we measured the cellular morphology, cell viability and membrane integrity as cytotoxicity indicators in HUVECs. Exposure to cytotoxic agents can affect cellular morphology, which is directly reflecting cell injuries. Firstly, we examined the morphology of HUVECs exposure to silica nanoparticles for 24 h by optical microscopy (). Cell density reduction, irregular shape as well as cellular shrinkage were observed. Changes in cellular morphology have been considered as a direct indicator in assessing cytotoxicity 
. To confirm and analyze this observation, cell viability and LDH release were measured (). Our data revealed that the silica nanoparticles-induced cytotoxicity increased in a dose- and time-dependent manner.
It has been confirmed that the LDH release is an indicator of necrosis due to cell membrane damage 
. To further analyze the cell death caused by silica nanoparticles, apoptosis and necrosis in HUVECs were measured (). In accordance with LDH results, a significant increase of necrosis rate was noted at the concentrations (25, 50, 75, and 100 µg/mL) of silica nanoparticles, while the apoptosis rate was much lower than necrosis (). Rapid-acting metabolic poisons and strong physical stress can cause necrosis accompanied with membrane damage. In contrast, apoptosis is a slow-acting form of cell death accompanied with an energy-dependent sequence of events, resulted in fragmenting nuclei and cytoplasmic organelles ultimately, thus, the membrane damage is not a primary event of apoptosis 
. In this study, we found that HUVECs exposure to silica nanoparticles also caused apoptosis (). Similar results were obtained from Liu and coworker who suggested that the endothelial cells exposure to silica nanoparticles could cause apoptosis 
. Endothelial cells apoptosis was considered as a major determinant of atherothrombosis 
. To investigate the possible mechanisms of apoptosis induced by silica nanoparticles, intracellular ROS, MDA and antioxidant activities including SOD and GSH-Px were measured (). The generation of intracellular ROS caused oxidative damage followed the production of lipid peroxidation and the inhibition of antioxidant activities. Generally, the oxidative stress produced by nanoparticles was considered to be one of the important aspects associated with nanotoxicity 
. It was reported that silica nanoparticles showed stable surface radicals and sustained release of Hydroxyl radical (·OH). The ·OH radical is the most reactive ROS and triggers extensive cellular damage. ROS generated by the silica nanoparticles surface can induce cell membrane damage via lipid peroxidation that may subsequently lead to increased cellular permeability 
. Oxidative stress is the result of an imbalance in the pro-oxidant/antioxidant homeostasis. It is well-known that extensive increase in the ROS production exceeds the capacity of antioxidant mechanisms causing injury to lipids, proteins and DNA 
. Induction of oxidative stress by silica nanoparticles have been observed in various cell types 
. It may also due to their direct or indirect effects to some organelles of nanoparticles which entered the cells. These organelles, such as mitochondria, are the main sources of cellular ROS and the basis of the ROS metabolism 
. Oxidative stress induced membrane lipid peroxidation could occur both in vitro and in vivo, especially in membranes of highly metabolically active mitochondria 
. In the present study, the mitochondrial membrane potential decreased obviously in a dose-dependent manner (). Excess ROS production produced by silica nanoparticles exposure is one of the factors leading to the collapse of mitochondrial membrane potential 
. Since the maintenance of ROS homeostasis depended on the respiratory chain and the membrane potential, oxidative damage may occur due to the decreasing of membrane potential 
In addition, DNA damage could be mediated by oxidative stress depending on the balance between ROS production and antioxidant status 
. The high surface area associated with nanoparticles can promote the generation of ROS, resulting in oxidative DNA damage 
. Our previous study confirmed that silica nanoparticles induced ROS directly lead to DNA damage and cell cycle arrest 
. The cellular response to DNA damage, commonly known as DDR, encompasses multiple repair mechanisms and checkpoint responses that can delay cell cycle progressing or modulate DNA replication 
. It had been reported that silica nanoparticles could induce DDR, mutagenic effects and cell cycle arrest in various non-endothelial cell lines 
. However, whether the toxic effect of endothelial cells is associated with DDR pathways has not been reported. In the present study, our results showed that the degree of DNA damage including the percentage of tail DNA, tail length and Olive tail moment (OTM) were significantly aggravated in a dose-dependent manner ( and ). Moreover, our data indicated that the silica nanoparticles inhibited HUVECs proliferation by inducing G2/M arrest ( and ). In response to DNA damage, cells launch elegant networks of genome surveillance mechanisms, called cell cycle checkpoints, to detect and repair damaged DNA to maintain the genome stability 
. When cells have DNA damage to be repaired or DNA replication is not complete, these checkpoints will arrest cell cycle at one of the G0/G1, S or G2/M phase. The G2/M phase has played an important role in mitotic processes. G2/M DNA damage checkpoint serves to prevent the cell from entering mitosis (M-phase) with genomic DNA damage 
. This kind of cell cycle delay could offer more time for the repair of DNA damage and avoid gene mutation 
. However, when the DNA injuries of cells were so severe that exceed the cellular repair capacity, apoptosis would occur. Cell cycle checkpoints are pivotal mechanisms safeguarding genome stability. Cells that harbor defects in checkpoints are predisposed to genome instability and neoplastic transformation 
. Therefore, it is necessary to further investigate the cell signaling pathway of silica nanoparticles-induced G2/M arrest.
In the current study, we confirmed that silica nanopaticles triggered DDR pathways leading to activate the G2/M cell cycle checkpoint. As shown in , we found that Cdc25C, Cdc2 and cyclin B1 were remarkable suppressed in HUVECs after exposure to silica nanoparticles for 24 h, while Chk1 was significantly increased. Checkpoint kinase 1 (Chk1), which is an essential kinase required to preserve genome stability, is activated in response to DNA damage and is involved in the cell cycle checkpoint control, DNA damage repair and DNA damage-induced apoptosis 
. In particular, Chk1 is mainly responsible for the G2/M DNA damage checkpoint signal transduction pathway 
. Upon to DDR, Chk1 is activated and inhibits the activation of the downstream target of Cdc25C, resulted in the downregulation of cyclinB1/Cdc2 kinase 
. Cdc2 and cyclin B1 are essential for the entry of cells into mitosis. Cdc2 is inactive as a monomer and must bind with cyclin B1 during the G2/M transition. Inhibition of cyclin B1/Cdc2 complex resulted in a directly G2/M arrest 
. Thus, we could confirm that the mechanisms of silica nanoparticles induced endothelial cells toxic effect was through activating the Chk1-dependent G2/M DNA damage checkpoint signaling pathway. The molecular mechanism obtained from our study may add information to the epidemiologic data that exposure to ultrafine particles is a significant risk for the development of cardiovascular diseases.