Toxicology Studies Of Typical Nanomaterials And Nanoformulation

This study explored toxic effects of nano-Fe₃O₄,carbon nanotube,nano-TiO₂ and nanoformulation in vitro and in vivo.Its main contents were as follows:First,studies on cytotoxicity of nanomaterials in A549 cells:The present study was to investigate the cytotoxicity effects of nano Fe₃O₄,different carbon nanotubes and nano-TiO₂ on human lung epithelial cell A549 in vitro.The change of the cell morphology was observed through inverted phase-contrast microscope,cell viability was determined by the MTT,CCK-8 and Trypan blue exclusion assays.Additionally,lactate dehydrogenase was determined in the medium supematant of A549 cells after treatment with different nanomaterials.The results showed that nano-Fe₃O₄ at the 100μg/ml had no obvious effects on cell viability,nano-TiO₂ at the 200μg/ml also had no obvious effects on cell viability.However,when cell toxicity of carbon nanotube was evaluated,we found that results of cell toxicity were not consistent among above methods.At the high concentration,CCK-8 method showed that cell viability was under 20%, however,cell viability had no obviously decrease under light microscope and LDH method and Trypan blue exclusion also demonstrated that carbon nanotube had no significant effects on cell viability.Taken together,nano Fe₃O₄,carbon nanotubes and nano-TiO₂ show no significant cytotoxicity in A549 cells.Conventional detecting methods of cytotoxicity can not be suitable to evaluate cytotoxicity of nanomaterials.Second,acute toxicity study of nanomaterials in ICR mice:The present study was to investigate the acute toxicity effects of nanomaterials by oral exposure in mice.The mice were treated by a single oral dose of nano-TiO₂ and single wall carbon nanotube at 5g/kg or multi-wall carbon nanotubes at 1g/kg body weight.After 14 days,the mice were sacrificed and the effects of body weight,food consumption,serum biochemistry,coefficients of organs and histopathology were measured.The results showed that nano-TiO₂ and carbon nanotubes had no obvious effects on body weight and coefficients of organs.Food consumption of the mice increased by gradually during the test.There were not obvious toxic reaction and death during observation.Although some serum biochemistry parameters had been significantly changed, these values were at the normal value except CK value which was in the male mice treated with nano-TiO₂,and there were not obvious changes by histopathology examination.Under the current conditions,nanomatierials were low toxicity or no toxicity.Third,subchronic toxicity study of nano-TiO₂ in SD rats:The present study was to explore subchronic toxicity study of nano-TiO₂ in SD rats,then observe toxic reaction and supply target organ of toxic reaction and its recovery.There were not any abnormal signs,toxic reaction and death in rats at the dose of 1g/kg during consecutive dosing 3 months.Compared with control group,there was no apparent change in food consumption and body weight.Hematology and serum biochemistry showed that some parameters had significant higher or lower than those of control group,but these values were at the normal values except BUN and CREA values in the female rats at the end of treatment with nano-TiO₂ and M value in the female rats at the end of treatment with micro-TiO₂,so changes of these values were not considered as treatment-related. Microscopy examination had no reveal any histopathology changes in all animals,there were not obvious changes of bone marrow cell morphology and pathology in bone marrow smears.Fourth,distribution study of nano-TiO₂ in SD rats:In the present study,we investigated distribution of nano-TiO₂ by ICP-MS method after a 13-week oral administration of nano-TiO₂ in SD rats.The samples included liver,spleen,kidney,intestine,brain and blood.The analysis of Ti concentration showed that nano-TiO₂ could be absorbed into circulation through gastrointestinal tract after nano-TiO₂ was administrated and its concentration was the most in the intestine,liver and spleen.The results revealed that nano-TiO₂ could be absorbed into circulation through gastrointestinal tract after nano-TiO₂ was administrated and distributed to tissue and organ,but it had no obvious effects on organism.Fifth,cutaneous toxicity of nanoformulation and its mechanism in beagle dogs:The cutaneous toxicity and toxicokinetics of nanoformulation were investigated in beagle dogs by intravenous administration once every 28 days for 12 weeks.We evaluated cutaneous toxicity once weekly in beagle dogs.The results showed that there was not cutaneous toxicity at control group,vehicle group and common formulation group.Cutaneous toxicity could be seen at the 0.1, 0.3 and 0.45mg/kg nanoformulation and there was a significant dose-dependence.Signs were mainly alopecia,erythema,exudation,eschar and ulcer in four limbs,jaw and ventral body.The results of toxicokinetics showed that pharmacokinetic of nanoformulation in beagle dogs was linear dynamics and had not to accumulate after repeated dosing.After changing formulation,drug could stay in plasma for a long time.For being eliminated quickly from plasma,common formulation almost could not be measured except for reaching peak concentration which was equal to 1/100 of nanoformulation at the same dose.Also it had been demonstrated that nanoformulation had a long circulation property in vivo.Analysis of concentration in the skin showed that drug concentration of ulcerate skin was higher than that of normal skin,it hinted that nanoformulation was easy to distribute to these places and resulted in cutaneous toxicity.Finally,the effects of nanomaterials on BBB model in vitro:Primary BCEC and astrocyte from rats were isolated and cultured and they were identified through cell morphology and immunohistochemistry.The results showed that cell purity was high,they could be used to establish BBB mode.Then,we established no-contact coculture model.Transendothelial electric resistance and permeability coefficient of fluorescein sodium were 373±41Ω·cm² and （0.34±0.14）×10^-3cm/min,respectively.These values were consistent with literature.Finally,we evaluated toxic effects of nanomaterials on tight junction of BBB.The results showed that these nanomaterials at the concentration of 100μg/ml had no obvious toxic effects.Taken together,under the current experiment condition,inorganic nanomaterials have no obvious toxic effects in vitro and in vivo.Biodegradable nanoformulation may lead to new toxicity which is different with common formulation because of distribution change. Conventional detecting methods of toxicity can not be suitable to evaluate toxicity of nanomaterials.