Cell-based Dual-fluorescent Biosensors For The Bioavailability Of Nanoparticles

Nanoparticles are particles with lengths that range from 1 to 100 nanometers intwo or three dimensions that may present a variety of hazards for human health.Thepresence of nanoparticles in commercially available products is becoming morecommon and developing for the application in coating,catalysts,semiconductor,cosmetics,food additive,electrical circuits,drug-delivery agents and so on.However,release of manufactured nanoparticles into the aquatic environment is largely possiblehazards associated with nanomaterials and harmful effects that may result fromexposure of aquatic animals to nanoparticles.So far,conventional methods are oftenadopted when detecting the toxicity of nanoparticles.In the context of particletoxicology,reporter cell based assays have proven useful in the assessment ofNP-derived immune responses.Here,a series of reporter plasmids have beenconstructed successfully which contain a mCherry gene regulated by an induciblepromoter?an activator protein 1?AP-1?promoter,an interleuk:in 8?IL8?promoter,ora B cell translocation gene 2?BTG2?promoter?,with an enhanced green-fluorescentprotein gene driven by the cytomegalovirus?CMV?promoter as the internal control.After optimizing flow cytometric analysis,these dual-fluorescence cell-basedbiosensors were capable of accurately and rapidly detecting NP toxicity.Theresponses of AP-1.BTG2,and IL8 biosensors in assessing the toxicity of silvernanoparticles?Ag NPs?were consistent with data acquired by conventional assays,such as western blot,real-time polymerase chain reaction,and immunofluorescence.Using this dual-fluorescence biosensor system,we investigated the toxicity ofnanoparticles and the influence of environmental factors?aging,fulvic acid?FA?,andUVA irradiation?on NP toxicity.Our results indicated that this novel cell-basedbiosensor system have proven useful in the assessment of expression profiling.Thecytoxicity and genotoxicity of TiO2 NPs were detected via additional biochemicalendpoints.The main results of this dissertation are listed below:1.This study developed a novel dual-fluorescence biosensor system based onthree inducible promoters?AP-1 promoter,IL8 promoter,or BTG2 promoter?.Byoptimizing the cell lines,cell culture and flow cytometric analysis,the accuracy andsensitivity of these biosensors were improved.Using these biosensors,the toxicity ofnanoparticles was detected rapidly and sensitively.and the results were consistent with those of conventional assays.2.The novel cell-based biosensor system constituted an accurate and sensitive method for rapidly evaluating NP toxicity and exhibited great potential for applications involving the assessment of toxic risk.Our results indicated that metal and metal oxide nanoparticles were generally considered to have toxicity in which AP-1 promoter,IL8 promoter,and BTG2 promoter were increased in a dose-dependentmanner,whereas carbon-based nanoparticles showed no significant increase in the AP-1 promoter response.3.The influence of environmental factors?aging,FA and UVA irradiation?on NP toxicity were investigated and aged particles are more toxic to cells as compared with these freshly prepared.FA administration reduced the particle toxicity.The influence of sunlight irradiation is changed according to the difference of the type and size of nanoparticles.4.Further evaluation of the toxicity of TiO₂ NPs,our results showed that TiO₂ NPs?5.15 nm?showed the low toxicity involved in cell viability.Exposure to 15-nm TiO2 NPs can increase the activity of AP-1 promoter,IL8 promoter,and BTG2 promoter involved in potential carcinogenesis,DNA damage,or inflammatory response,respectively.TiO2 NPs?15 nm?showed high genotoxicity on DNA damage which was repaired by the DNA repair system in accuracy.