Cytotoxicity Of Black Phosphorus Nanosheets In Vascular Endothelial Cells And Underlying Mechanisms

Background and ObjectiveBlack phosphorus(BP),as the most stable allotrope of phosphorus,has drawn attention from the academic community due to its graphene-like six-membered ring and layered structure at the microscopic level.As a novel two-dimensional nanomaterial composed of a single element,BP has found wide applications in the fields of mechanics,optics,and semiconductors,thanks to its unique structure and single-layer band gap.In the biomedical field,its single-layer corrugated shape provides opportunities for drug loading and functional modification.Currently,BP has been extensively studied in targeted therapy,photothermal therapy,and biosensors.As the degradation product of BP is non-toxic phosphate,it can provide the necessary substrate for bone formation.Although current research generally believes that BP nanomaterials have good biocompatibility,nano-materials inevitably enter the circulatory system after entering the body through various pathways,and there is not enough research on the toxic effects of BP on vascular tissues.This study aims to explore the toxic effects of BP nanomaterials on vascular tissues and their related mechanisms through in vitro experiments.Methods and MaterialsThis experiment prepared black phosphorus nanosheets(BPNSs)with an average particle size of less than 300 nm by the classic liquid phase exfoliation method.The surface physicochemical properties of the samples were detected by atomic force microscopy,scanning electron microscopy,transmission electron microscopy,Raman spectroscopy,and dynamic light scattering.After dilution,BPNSs were co-cultured with human umbilical vein endothelial cells(HUVECs),and cell uptake detection,cell time-lapse imaging,cell proliferation experiments,cell skeleton staining,cell scratch experiments,cell membrane integrity detection,intracellular reactive oxygen species detection,mitochondrial membrane potential detection,cell apoptosis detection,and real-time fluorescence quantitative polymerase chain reaction were used to experimentally analyze cell morphology,cell viability,internal cell structure,cell migration function,cell membrane integrity,intracellular oxidative stress,cell apoptosis levels,and apoptosis-related gene expression levels.ResultsThe BPNSs prepared by liquid phase exfoliation meet the experimental requirements in terms of size,structure,and dispersibility.BPNSs were taken up by HUVECs shortly after co-culture,and the cytotoxicity of BPNSs was positively correlated with concentration and time.When the concentration exceeded 2.5 μg/mL after 24 hours of culture,BPNSs affected the cell skeleton structure and inhibited cell migration.BPNSs disrupted cell membrane integrity,increased intracellular reactive oxygen species levels,promoted mitochondrial membrane potential reduction,and facilitated cell apoptosis.The expression level of P53 increased,while the expression levels of BCL-2 decreased,and the expression levels of BAX increased within the cells.ConclusionsBPNSs showed concentration-dependent and time-dependent cytotoxicity to HUVECs,with significant toxicity observed at concentrations exceeding 2.5 μg/mL.The cytotoxicity induced by BPNSs was attributed to the disruption of cell membranes,oxidative stress,mitochondrial dysfunction,promotion of cell apoptosis-related gene expression,leading to cell apoptosis and affecting cell vitality and function.This study clarified the role and regulatory mechanisms of reactive oxygen species and apoptosisrelated genes in the BPNSs-induced cytotoxicity,providing ideas for a comprehensive understanding of the toxicity of BPNSs and further research based on BP.