Pamam Nanomaterials And Carbon Nanotubes Toxicity Studies

PAMAM dendrimer(polyamidoamine dendrimer) is a polymer with dentritic branching and radial symmetry.It consists of three parts:a central core,an interior dendritic structure(the branches) and an exterior suface(the end group).PAMAM dendrimers can be divided in to the whole(cationic) or half (anionic) generation polymers.The surface of the whole generation is functionalized with primary amino surface groups and the half generation is carboxylate surface group.Due to the cationic surface,the whole generation PAMAM could be used for gene delivery for expression in cells or in animals. Therefore,the biosafety of these PAMAMs need to be determined.In this dissertation,we determine the cell viability after PAMAM G1 to G8 and G3.5,G4.5,G5.5 and G7.5 treatment.We find that treatment of A549 by cationic PAMAM,from G3 to G8 could cause cell death,while the anionic PAMAMs have no effect on cell viability.Furthermore,we find that PAMAM G3 causes A549 cell death through autophagy,but not through apoptosis.Treating cells with the autophagy inhibitor,3-methyladenine,or the knockdown of the autophagy related gene,beclin,could improve the cell viability treated by PAMAM G3.In the meanwhile,autophagic cell death happens in the PAMAM G4,G5,G6,G7 and G8 treated A549 cells.In the following step,we analyze the signal transduction pathways in the PAMAM G3 induced autphogic cell death.We find that PAMAM G3 induced A549 autophagic cell death through AKT-TSC2-mTOR pathway. Furthermore,we discover that PAMAM G3 could induce acute lung injury and cause death in mice,while the autophagy inhibitor 3MA could improve the situation.It is indicated that autophagy plays an important role in PAMAM G3 induced mice acute lung injury.COOH functionalized single walled carbon nanotube induces A549 autophagic cell deathCarbon NanoTube(CNT) is a special form of carbon in which chemical bonds of carbon form tubes form carbon atoms.Usually there are two kinds of CNT:single-walled carbon nanotube(SWCNT),containing only one tube in the CNT and multi-walled carbon nanotube(MWCNT),containing more than one concentric tube in the basic element of the CNT.The CNTs may have different properties with different surface functionalizations.In this dissertation,we find COOH functionalized SWCNT induces A549 autophagic cell death,while the autophagy inhibitor 3MA could improve the situation.Furthermore,we discover COOH functionalized SWCNT downregulates p-AKT/AKT and p-mTOR/mTOR ratio in A549 cells,similar with PAMAM G3 treated A549 cells.The research on the COOH functionalized SWCNT induced autophagy is still under way,both in cell and mice.MWCNT induces A549 cell apoptosis in a caspase-3 independent pathwayWe find that MWCNT could induce A549 apoptosis.But it fails to active the caspase-3 in A549 cell.In the meanwhile,a pancaspase inhibitor,Z-VAD-fmk could not rescue the MWCNT induced apoptosis,indicating that MWCNT induced A549 cell apoptosis is in a caspase-3 independent pathway. Spike expression induces ER stress and interferes with insulin signal transduction pathway in HEK 293T ceilsEndoplasmic Reticulum is the place where secretary and transmembrane proteins are properly folded and decorated.Too much unfolded or misfolded protein will induce ER stress,in which situation cells will take unfolded protein response to elimate those proteins.It is reported that the spike expression could induce ER stress in HEK 293 cells.In this dissertation,we find that it is S2 (S681-1190) that induces ER stress in HEK 293T cells.Furtermore,we find that expression of S2 interferes with insulin signal transduction pathway,with downregulation of phospho-AKT/total AKT ratio.We speculate that S2 induced ER stress could interfere with insulin signaling,and the related research is going on.Endotoxin removal from the expressed spike protein and analysis of inflammatory cytokines induced by spike proteinSARS-CoV infection could induce severe inflammatory reaction,with a elevation of inflammatory cytokine in the victims.We expresse and purify SARS-CoV spike protein in human cell line andfind that it could induce TNF-a, IL-1β,IL-6 and IFN-γsecretion in human monocytes from peripheral blood mononuclear cells.With the endotoxin detection test,we find there is a lot of endotoxin in the expressed spike protein.After endotoxin removal with endotoxin removal column, we find the spike protein could also induce TNF-a and IL-6 secretion,but to a much less degree compared with the previous protein before endotoxin removal. In the meanwhile,we could not detection IFN-γsecretion treated by spike protein after endotoxin removal.