title: Oxidative stress mediates the effects of Raman-active gold nanoparticles in human cells.
abstract: Polyethylene glycol (PEG)ylated Raman-active gold nanoparticles (PEG-R-AuNPs) consist of an interchangeable Raman organic molecule layer held onto a gold nanocore by a silica shell. PEG-R-AuNPs have been shown preclinically to increase the sensitivity and specificity of Raman spectroscopy, with picomolar sensitivity and multiplexing capabilities. Although clinical trials are being designed to use functionalized PEG-R-AuNPs in various applications (e.g., to target dysplastic bowel lesions during colonoscopy), the effects of these nanoparticles on human cells remain unknown. The occurrence and mechanisms underlying any potential cytotoxicity induced by these nanoparticles (0-1000 PEG-R-AuNPs/cell) are investigated in immortalized human HeLa and HepG2 cell lines at several time points (0-48 h) after exposure. Using fluorometric assays, cell viability (MTT), reactive oxygen species (ROS) generation (dichlorofluorescein diacetate), protein oxidation (protein carbonyl content), and total cellular antioxidant concentrations the concentrations (metmyoblobin-induced oxidation of ABTS) are assessed. Analysis of lipid oxidation using an enzyme immunoassay (8-isoprostane concentrations), gene expression of antioxidant enzymes using quantitative reverse transcription polymerase chain reactions, and the intracellular location of PEG-R-AuNPs using transmission electron microscopy is also undertaken. PEG-R-AuNPs cause no cytotoxicity in either HeLa or HepG2 cells in the acute setting as ROS generation is balanced by antioxidant enzyme upregulation. Following prolonged exposures (48 h) at relatively high concentrations (1000 PEG-R-AuNPs/cell), nanoparticles are found within vesicles inside cells. Under these conditions, a minimal amount of cytotoxicity is seen in both cell lines owing to increases in cellular oxidative stress, most likely due to ROS overwhelming the antioxidant defenses. Evidence of oxidative stress-induced damage includes increased lipid and protein oxidation. Although further in vivo toxicity studies are necessary, these initial encouraging results show that PEG-R-AuNPs cause minimal toxicity in human cells in the acute setting, which bodes well for potential future applications of these nanoparticles in living subjects.
authors.affiliation: Molecular Imaging Program at Stanford, Department of Radiology, Stanford University, The James H. Clark Center, 318 Campus Drive, Stanford, CA 94305-5427, USA.
authors.forename: Avnesh S
authors.initials: AS
authors.lastname: Thakor
authors.forename: Ramasamy
authors.initials: R
authors.lastname: Paulmurugan
authors.forename: Paul
authors.initials: P
authors.lastname: Kempen
authors.forename: Cristina
authors.initials: C
authors.lastname: Zavaleta
authors.forename: Robert
authors.initials: R
authors.lastname: Sinclair
authors.forename: Tarik F
authors.initials: TF
authors.lastname: Massoud
authors.forename: Sanjiv S
authors.initials: SS
authors.lastname: Gambhir
mesh_terms: D000975:Antioxidants / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D006046:Gold / Q000737:chemistry* / Q000494:pharmacology; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry*; D018384:Oxidative Stress / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000975:Antioxidants; D017382:Reactive Oxygen Species; D006046:Gold