title: Flotillin-involved uptake of silica nanoparticles and responses of an alveolar-capillary barrier in vitro.
abstract: Drug and gene delivery via nanoparticles across biological barriers such as the alveolar-capillary barrier of the lung constitutes an interesting and increasingly relevant field in nanomedicine. Nevertheless, potential hazardous effects of nanoparticles (NPs) as well as their cellular and systemic fate should be thoroughly examined. Hence, this study was designed to evaluate the effects of amorphous silica NPs (Sicastar) and (poly)organosiloxane NPs (AmOrSil) on the viability and the inflammatory response as well as on the cellular uptake mechanisms and fate in cells of the alveolar barrier. For this purpose, the alveolar epithelial cell line (NCI H441) and microvascular endothelial cell line (ISO-HAS-1) were used in an experimental set up resembling the alveolar-capillary barrier of the lung. In terms of IL-8 and sICAM Sicastar resulted in harmful effects at higher concentrations (60 μg/ml) in conventional monocultures but not in the coculture, whereas AmOrSil showed no significant effects. Immunofluorescence counterstaining of endosomal structures in NP-incubated cells showed no evidence for a clathrin- or caveolae-mediated uptake mechanism. However, NPs were enclosed in flotillin-1 and -2 marked vesicles in both cell types. Flotillins appear to play a role in cellular uptake or trafficking mechanisms of NPs and are discussed as indicators for clathrin- or caveolae-independent uptake mechanisms. In addition, we examined the transport of NPs across this in vitro model of the alveolar-capillary barrier forming a tight barrier with a transepithelial electrical resistance of 560±8 Ω cm(2). H441 in coculture with endothelial cells took up much less NPs compared to monocultures. Moreover, coculturing prevented the transport of NP from the epithelial compartment to the endothelial layer on the bottom of the filter insert. This supports the relevance of coculture models, which favour a differentiated and polarised epithelial layer as in vitro test systems for nanoparticle uptake.
authors.affiliation: Institute of Pathology, University Medical Centre, Mainz, Germany. kasperj@uni-mainz.de
authors.forename: Jennifer
authors.initials: J
authors.lastname: Kasper
authors.forename: Maria I
authors.initials: MI
authors.lastname: Hermanns
authors.forename: Christoph
authors.initials: C
authors.lastname: Bantz
authors.forename: Stefanie
authors.initials: S
authors.lastname: Utech
authors.forename: Olga
authors.initials: O
authors.lastname: Koshkina
authors.forename: Michael
authors.initials: M
authors.lastname: Maskos
authors.forename: Christoph
authors.initials: C
authors.lastname: Brochhausen
authors.forename: Christine
authors.initials: C
authors.lastname: Pohl
authors.forename: Sabine
authors.initials: S
authors.lastname: Fuchs
authors.forename: Ronald E
authors.initials: RE
authors.lastname: Unger
authors.forename: C James
authors.initials: CJ
authors.lastname: Kirkpatrick
mesh_terms: D002196:Capillaries / Q000187:drug effects*; D002460:Cell Line; D002470:Cell Survival; D018920:Coculture Techniques; D004396:Coloring Agents / Q000737:chemistry; D016503:Drug Delivery Systems; D017097:Electric Impedance; D004705:Endocytosis; D042783:Endothelial Cells / Q000187:drug effects; D006801:Humans; D007249:Inflammation; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000737:chemistry*; D008833:Microcirculation / Q000187:drug effects; D050997:Nanomedicine; D053758:Nanoparticles / Q000737:chemistry*; D011650:Pulmonary Alveoli / Q000187:drug effects*; D012235:Rhodamines / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry*
chemical_list: D004396:Coloring Agents; D008051:Lipid Bilayers; D008565:Membrane Proteins; D012235:Rhodamines; C106354:flotillins; D012822:Silicon Dioxide; C029773:rhodamine B