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Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods.

ABSTRACT: Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of super-paramagnetic iron oxide nanoparticles (SPIONs), safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity. Thus, treatment with lauric acid-coated SPIONs (SEON(LA)) resulted in a significant increase in the intensity of side scatter and toxicity, whereas SEON(LA) with an additional protein corona formed by bovine serum albumin (SEON(LA-BSA)) and commercially available Rienso(®) particles showed only a minimal increase in both side scatter intensity and cellular toxicity. The increase in side scatter was in accordance with the measurements for SPION content by the atomic adsorption spectroscopy reference method. In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products.

SUBMITTER: Friedrich RP

PROVIDER: S-EPMC4492632 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods.

Friedrich Ralf P RP Janko Christina C Poettler Marina M Tripal Philipp P Zaloga Jan J Cicha Iwona I Dürr Stephan S Nowak Johannes J Odenbach Stefan S Slabu Ioana I Liebl Maik M Trahms Lutz L Stapf Marcus M Hilger Ingrid I Lyer Stefan S Alexiou Christoph C

International journal of nanomedicine 20150626

Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of super-paramagnetic iron oxide nanoparticles (SPIONs), safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantifi ...[more]

PMID: 26170658

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Project description:BackgroundNucleic acids can fold into non-canonical secondary structures named G-quadruplexes (G4s), which consist of guanine-rich sequences stacked into guanine tetrads stabilized by Hoogsteen hydrogen bonding, ?-? interactions, and monovalent cations. G4 structure formation and properties are well established in vitro, but potential in vivo functions remain controversial. G4s are evolutionarily enriched at distinct, functional genomic loci, and both genetic and molecular findings indicate that G4s are involved in multiple aspects of cellular homeostasis. In order to gain a deeper understanding of the function of G4 structures and the trigger signals for their formation, robust biochemical methods are needed to detect and quantify G4 structures in living cells. Currently available methods mostly rely on fluorescence microscopy or deep sequencing of immunoprecipitated DNA or RNA using G4-specific antibodies. These methods provide a clear picture of the cellular or genomic localization of G4 structures but are very time-consuming. Here, we assembled a novel protocol that uses the G4-specific antibody BG4 to quantify G4 structures by flow cytometry (BG-flow).ResultsWe describe and validate a flow cytometry-based protocol for quantifying G4 levels by using the G4-specific antibody BG4 to label standard cultured cells (Hela and THP-1) as well as primary cells obtained from human blood (peripheral blood mononuclear cells (PBMCs)). We additionally determined changes in G4 levels during the cell cycle in immortalized MCF-7 cells, and validated changes previously observed in G4 levels by treating mouse macrophages with the G4-stabilizing agent pyridostatin (PDS).ConclusionWe provide mechanistic proof that BG-flow is working in different kinds of cells ranging from mouse to humans. We propose that BG-flow can be combined with additional antibodies for cell surface markers to determine G4 structures in subpopulations of cells, which will be beneficial to address the relevance and consequences of G4 structures in mixed cell populations. This will support ongoing research that discusses G4 structures as a novel diagnostic tool.

Dataset Information

Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods.

Publications

Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods.

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