title: Nonendosomal cellular uptake of ligand-free, positively charged gold nanoparticles.
abstract: Gold nanoparticles (GNPs) have interesting optical properties, such as exceptionally high quantum yields and virtually limitless photostability. Therefore, they show the potential for applications as biomarkers especially suitable for in vivo and long-term studies. The generation of GNPs using pulsed laser light rather than chemical means provides nanoparticles, which are remarkably stable in a variety of media without the need of stabilizing agents or ligands. This stabilization is achieved by partial oxidation of the gold surface resulting in positively charged GNPs. However, little is known about cellular uptake of such ligand-free nanoparticles, their intracellular fate, or cell viability after nanoparticle contact. The current work is aimed to explore the response of a bovine cell line to GNP exposure mainly using laser scanning confocal microscopy (LSCM) supported by other techniques. Cultured bovine immortalized cells (GM7373) were coincubated with GNP (average diameter 15 nm, 50 microM Au) for 2, 24, and 48 h. The detection of GNP-associated light scattering by the LSCM facilitated a clear distinction between GNP-containing cells and the negative controls. After 48 h, 75% of cells had visibly incorporated nanoparticles. No colocalization was detected with either Rab5a or Lamp1-positive structures, i.e., endosomes or lysosomes, respectivley. However, transmission electron microscope analysis of GNP-coincubated cells indicated the nanoparticles to be positioned within electron-dense structures. Coincubation at 4 degrees C did not inhibit nanoparticle uptake, suggesting diffusion as possible entrance mechanism. Although the assessment of cell morphology, membrane integrity, and apoptosis revealed no GNP-related loss of cell viability at a gold concentration of 25 microM or below, a cytotoxic effect was observed in a proliferation assay after exposing low cell numbers to 50 microM Au and above. In conclusion, this study confirmed the cellular uptake of ligand-free gold nanoparticles during coincubation apparently without using endocytic pathways.
authors.affiliation: Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Federal Research Institute of Animal Health, Mariensee, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Taylor
authors.forename: Sabine
authors.initials: S
authors.lastname: Klein
authors.forename: Svea
authors.initials: S
authors.lastname: Petersen
authors.forename: Wilfried
authors.initials: W
authors.lastname: Kues
authors.forename: Stephan
authors.initials: S
authors.lastname: Barcikowski
authors.forename: Detlef
authors.initials: D
authors.lastname: Rath
mesh_terms: D000818:Animals; D001692:Biological Transport; D002417:Cattle; D002460:Cell Line; D002470:Cell Survival; D011992:Endosomes / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D006046:Gold / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007834:Lasers; D008024:Ligands; D053768:Metal Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D018613:Microscopy, Confocal; D011419:Propidium / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013997:Time Factors
chemical_list: D008024:Ligands; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011419:Propidium; D006046:Gold