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Jahresbericht 2014 - Annual Report 2014

56 QUANTIFICATION OF INTERNALIZED SILICA NANOPARTICLES VIA STED MICROSCOPY In nanosafety, dosimetry is an important parameter for the under- standing of nanoparticle (NP) induced eff ects on a cellular scale as well as for risk assessment. Not only in vivo but also for in vitro experi- ments, it is critical to have information about the administered NP dose initially added, the delivered dose, comprising the particles reaching the cells, and the intracellular dose, internalized by the cells (Fig. 1). When comparing various NP sizes, it is important to recognize that at equal mass concentrations larger numbers of smaller NPs are present. Th erefore, appropriate analyses of NP internalization effi ciencies need to consider other measures, i.e. NP number concentrations. In this study, the internalization of 25 nm and 85 nm silica NPs in A549 cells, a model for type II alveolar epithelial cells, was quantifi ed. Cells were exposed to equal initial NP number concentrations (9.2x1010 NPs ml-1 ) of each particle size. Sedimentation was calculated to cause an increase in the NP number concentration immediately surrounding the cells (2.3x1012 NPs ml-1 , delivered dose). Th e number of internalized NPs was extracted from 3D super-resolution STED (stimulated emission depletion) image stacks of entire cells (Fig. 2) via image processing. STED images revealed that particles of both sizes entered the cells aft er 5 h incubation in serum supplemented medium. Th e particles appeared to be distributed throughout the cells, but were not detected in the nucleus. Taking the formation of small agglomerates that could not be resolved by STED in case of the 25 nm NPs into ac- count, the number of internalized NPs was estimated to be in the same range (2.5 1011 -4.8 1012 NPs ml-1 cell volume) at both particle sizes, with the 25 nm NPs only slightly stronger accumulated. Th e intracellular NP concentration did not signifi cantly exceed the delivered NP dose within 5 h. Th e experiments were performed at non-cytotoxic NP concentra- tions, excluding an infl uence of cytotoxicity on NP uptake. HIGHLIGHTS T. RUCKELSHAUSEN, H. PEUSCHEL, C. CAVELIUS, A. KRAEGELOH Fig. 1: Measures in NP dosimetry. Fig. 2: Image of a single A549 cell (cell membrane: cyan, nuclear membrane: yellow, confocal) after exposition to 85 nm-silica NPs (magenta, STED). A) 2D section (xy) and B) orthogonal section (xz). 2 µm B xz A xy NANO CELL INTERACTIONS range (2.51011 -4.81012

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