Kontrollierte Deposition synthetischer Nanopartikel auf Lungenzellen an der Luft-Flüssigkeits-Grenzschicht für toxikologische Untersuchungen

In der Dosimetrie und Charakterisierung relevanter Materialeigenschaften stoßen etablierte Expositionsverfahren zur Bewertung der toxischen Wirkung von nanoskaligen Partikeln an ihre Grenzen. Zur Untersuchung der Wirkung inhalierter Partikel wird daher zunehmend das Verfahren zur in vitro Exposition von Lungenzellen an der Luft-Flüssigkeits-Grenzschicht eingesetzt. In dieser Arbeit wurde ein entsprechendes Expositionssystem aufgebaut, im Detail charakterisiert und für die Untersuchung von Nanopartikeln eingesetzt. Zur Vorhersage der Depositionsezienzen wurde ein uid-dynamisches Modell für Partikelgröÿen zwischen 20 nm und 2 m entwickelt und experimentell validiert. Die relativ geringe Depositionsezienz der Expositionskammern von ca. 1% wurde durch ein elektrostatisches Feld um mehr als eine Gröÿenordnung erhöht, so dass die Simulation akut erhöhter Partikelbelastungen möglich ist. Die Funktionstüchtigkeit des Expositionssystems wurde mit luftgetragenen Metalloxidnanopartikeln (TiO2, SiO2) und Ruÿaerosol demonstriert. Dazu wurden verschiedene Aerosolerzeugungsverfahren getestet und die Aerosole insbesondere bezüglich ihres Agglomerationszustands und Residualpartikelanteils charakterisiert. Zur Bestimmung der deponierten Dosis wurden verschiedene Methoden untersucht und deren Anwendungsbereiche eingegrenzt. Die aus den Expositionsversuchen ermittelten wirksamen Dosen wurden mit realistischen Partikelbelastungen verglichen und den beobachteten Reaktionen der A549 Lungenzelllinie gegenübergestellt. Akute inammatorische und zytotoxische Zellreaktionen wurden nur nach Exposition mit unnatürlich hohen Dosen an SiO2 Nanopartikeln beobachtet

Silica nanoparticles are less toxic to human lung cells when deposited at the air-liquid interface compared to conventional submerged exposure

Background: Investigations on adverse biological effects of nanoparticles (NPs) in the lung by in vitro studies are usually performed under submerged conditions where NPs are suspended in cell culture media. However, the behaviour of nanoparticles such as agglomeration and sedimentation in such complex suspensions is difficult to control and hence the deposited cellular dose often remains unknown. Moreover, the cellular responses to NPs under submerged culture conditions might differ from those observed at physiological settings at the air–liquid interface.Results: In order to avoid problems because of an altered behaviour of the nanoparticles in cell culture medium and to mimic a more realistic situation relevant for inhalation, human A549 lung epithelial cells were exposed to aerosols at the air–liquid interphase (ALI) by using the ALI deposition apparatus (ALIDA). The application of an electrostatic field allowed for particle deposition efficiencies that were higher by a factor of more than 20 compared to the unmodified VITROCELL deposition system. We studied two different amorphous silica nanoparticles (particles produced by flame synthesis and particles produced in suspension by the Stöber method). Aerosols with well-defined particle sizes and concentrations were generated by using a commercial electrospray generator or an atomizer. Only the electrospray method allowed for the generation of an aerosol containing monodisperse NPs. However, the deposited mass and surface dose of the particles was too low to induce cellular responses. Therefore, we generated the aerosol with an atomizer which supplied agglomerates and thus allowed a particle deposition with a three orders of magnitude higher mass and of surface doses on lung cells that induced significant biological effects. The deposited dose was estimated and independently validated by measurements using either transmission electron microscopy or, in case of labelled NPs, by fluorescence analyses. Surprisingly, cells exposed at the ALI were less sensitive to silica NPs as evidenced by reduced cytotoxicity and inflammatory responses.Conclusion: Amorphous silica NPs induced qualitatively similar cellular responses under submerged conditions and at the ALI. However, submerged exposure to NPs triggers stronger effects at much lower cellular doses. Hence, more studies are warranted to decipher whether cells at the ALI are in general less vulnerable to NPs or specific NPs show different activities dependent on the exposure method

Silica nanoparticles are less toxic to human lung cells when deposited at the air–liquid interface compared to conventional submerged exposure

Background: Investigations on adverse biological effects of nanoparticles (NPs) in the lung by in vitro studies are usually performed under submerged conditions where NPs are suspended in cell culture media. However, the behaviour of nanoparticles such as agglomeration and sedimentation in such complex suspensions is difficult to control and hence the deposited cellular dose often remains unknown. Moreover, the cellular responses to NPs under submerged culture conditions might differ from those observed at physiological settings at the air–liquid interface.Results: In order to avoid problems because of an altered behaviour of the nanoparticles in cell culture medium and to mimic a more realistic situation relevant for inhalation, human A549 lung epithelial cells were exposed to aerosols at the air–liquid interphase (ALI) by using the ALI deposition apparatus (ALIDA). The application of an electrostatic field allowed for particle deposition efficiencies that were higher by a factor of more than 20 compared to the unmodified VITROCELL deposition system. We studied two different amorphous silica nanoparticles (particles produced by flame synthesis and particles produced in suspension by the Stöber method). Aerosols with well-defined particle sizes and concentrations were generated by using a commercial electrospray generator or an atomizer. Only the electrospray method allowed for the generation of an aerosol containing monodisperse NPs. However, the deposited mass and surface dose of the particles was too low to induce cellular responses. Therefore, we generated the aerosol with an atomizer which supplied agglomerates and thus allowed a particle deposition with a three orders of magnitude higher mass and of surface doses on lung cells that induced significant biological effects. The deposited dose was estimated and independently validated by measurements using either transmission electron microscopy or, in case of labelled NPs, by fluorescence analyses. Surprisingly, cells exposed at the ALI were less sensitive to silica NPs as evidenced by reduced cytotoxicity and inflammatory responses.Conclusion: Amorphous silica NPs induced qualitatively similar cellular responses under submerged conditions and at the ALI. However, submerged exposure to NPs triggers stronger effects at much lower cellular doses. Hence, more studies are warranted to decipher whether cells at the ALI are in general less vulnerable to NPs or specific NPs show different activities dependent on the exposure method