Study Of Synergistic Pulmonary Toxicity Of Nano Carbon Black-metal Ions Co-exposure

Particulate matter(PM)in the air is the principal component of air pollution that pose a threat to human health.Currently,a number of studies have investigated the toxicity of PM.However,the toxic effects mechanism caused by PM is still not well studied.As the sedimentation and accumulation of small PM are faster than the large PM that exist in the air,then cause a greater threat to human health.Therefore,current research on threats of PM 2.5 to human health is more extensive.It is considered that the interactions between chemicals adsorbed on PM 2.5 and PM 2.5 has a significant effect on the toxicity of PM.Similar to nanoparticles,PM 2.5,with large surface area to volume ratio,can absorb multipollutants in air,displaying toxicity profiles that are very different from those of coarse particles of the same composition.One particularly relevant interaction is that of PM 2.5 and the anthropogenic metals.From another point of view,we consider that investigating the biological outcomes of nanomaterials with sub-micro nanometers is a good approach to understand the PM 2.5 toxicity mechanism.CBs in air mainly come from the ash produced by combustion sources.As a type of pure carbon particles,CBs have been used extensively in toxicology studies as a model for airborne PM.In this study,we used CBs and metal ions as model materials to investigate the cytotoxicity of CBs and synergistic cytotoxicity and pulmonary toxicity of CBs and metal ions and its mechanism.The main results are described as following:Firstly,we investigated the cytotoxic effects of CBs with different sizes(14,51 and 95 nm)on mouse macrophage RAW264.7 cells.We found the dose-dependent and size-dependent effect of cellular uptake and cytotoxicity of CBs.Oxidative stress and nuclear damage induced by cellular uptake of a large number of CBs is a key factor in the cytotoxicity of CBs.Secondly,we investigated the co-cytotoxicity of CBs and different metal ions.We found the synergistic cytotoxicity of CBs and metal ions to RAW264.7 cells and BEAS-2B cells.The adsorption of CBs and metal ions and the distribution of CBs-metal complexes in the cells were measured using synchrotron radiation-based X-ray imaging and analysis techniques.The results showed that CBs can adsorb and deliver metal ions into cells,resulting in increased intracellular metal concentration,causing increased cytotoxicity.We investigated the mechanism of CBs-metal complex cytotoxicity.The autophagy and lysosomal dysfunction caused by CBs-metal complexes is the mechanism synergistic cytotoxicity of CBs and metal ions.Thirdly,the co-pulmonary toxicity of CBs and metal ions in vivo was studied.CBs and metal ions caused significant synergistic pulmonary toxicity.Analysis of the biochemical markers in the lung lavage fluid(BAL),as well as the inflammatory factors in serum and BAL,showed that CBs-metal ions co-exposure resulted in significant synergistic pulmonary toxicity.Pulmonary pathology study showed that CBs-metal ions co-exposure caused inflammatory lung infiltration,bronchial swelling,and bronchial wall thickening.ICP-OES and synchrotron radiation-based X-ray fluorescence microscopy(XRF)results showed that CBs delivered metal ions into lung,resulted in a sharp increase in the content of metal ions in lung.We found the autophagy and lysosomal dysfunction caused by CBs-metal ions co-exposure accounted for their synergistic pulmonary toxicity.Fouthly,DNA is not only the genetic material for coding,storing and transferring biological information,but also a versatile material for the “bottom-up” construction of exquisite nanostructures.There have been great advances in assembling a variety of complicated two-and three-dimensional DNA nanoarchitectures.Today,these DNA nanoarchitectures with excellent mechanical rigidity and structural stability have demonstrated great potential in a wide range of applications,including molecular sensoring,computation,nanomachines as well as diagnostics and therapeutics.Among various biological and biomedical applications,the interaction of DNA nanostructures with living systems and their biocompatibility are important issue.Current understanding of the toxicological of DNA nanostructures is still limited.Further systematic biocompatibility assessment of DNA nanostructure to various cell lines is essential.In this work,we choose tetrahedral DNA nanostructures(TDNs),one of the most practical DNA nanoconstructs,self-assembled simply from four DNA strands and prepared in a high yield,as a representative of DNA nanostructures.Normal bronchial epithelial cells(BEAS-2B cells),carcinoma cells(HeLa cells),and macrophage(RAW264.7 cells),which are widely used in biomedical research,were chosen as representative mammalian cell models.We observed different uptake dynamics of TDNs with those cell lines,demonstrated excellent innate biocompatibility of TDNs through cytotoxicity determination,protein expression assay,inflammation and apoptosis monitoring as well as cell organelle function detection.Significantly,we found that TDNs did not alter the cell cycle progression and cell division.All of the findings will deepen our understanding of the interaction of DNA nanostructures with mammalian cells,which will be useful for better designing DNA nanostructure-based bioprobes and biovectors for further applications.In conclusion,we investigated the cytotoxicity of CBs and synergistic pulmonary toxicity of CBs and metal ions and its mechanism.Results showed the size-dependent cellular uptake and cytotoxicity of CBs.We found co-exposure to CBs and metal ions caused a synergistic toxic effect.More importantly,we found that co-exposure to CBs and metal ions led to autophagy and lysosomal dysfunction,which accounted for the synergistic toxic effect of them.Our findings provide a new insight into understanding the toxicological and healthy effects of fine particles,which have potential to aid in developing pharmaceutical agents that target autophagy to decrease fine particle related respiratory infections and inflammation.Additionally,we demonstrated the good innate biocompatibility of TDNs in three types of cells through systematic biocompatibility assessment.Our findings not only offer new insights into the interaction of DNA nanostructures with mammalian cells,but will also be significant toward design of safe and effective DNA nanostructure-based bioprobes and biovectors for further applications.