Validation of weak biological effects by round robin experiments: cytotoxicity/biocompatibility of SiO2 and polymer nanoparticles in HepG2 cells

Lisa Landgraf,Peter Schmiel,Volker Mailänder,Christina Graf,Julia S Gebauer,Qi Gao,Reinhard Zellner,Carsten Weiss,Lennart Treuel,Ingrid Hilger,Stefan Graß,Daniel Nordmeyer,Katharina Landfester,Sandra Ritz,Eckart Rühl,Silvia Diabaté

doi:10.1038/s41598-017-02958-9

Abstract

All over the world, different types of nanomaterials with a diversified spectrum of applications are designed and developed, especially in the field of nanomedicine. The great variety of nanoparticles (NPs), in vitro test systems and cell lines led to a vast amount of publications with conflicting data. To identify the decisive principles of these variabilities, we conducted an intercomparison study of collaborating laboratories within the German DFG Priority Program SPP1313, using well-defined experimental parameters and well-characterized NPs. The participants analyzed the in vitro biocompatibility of silica and polymer NPs on human hepatoma HepG2 cells. Nanoparticle mediated effects on cell metabolism, internalization, and inflammation were measured. All laboratories showed that both nanoparticle formulations were internalized and had a low cytotoxicity profile. Interestingly, small variations in nanoparticle preparation, cell handling and the type of culture slide influenced the nanoparticle stability and the outcomes of cell assays. The round robin test demonstrated the importance of the use of clearly defined and characterized NPs and parameters for reproducible results across laboratories. Comparative analyses of in vitro screening methods performed in multiple laboratories are absolutely essential to establish robust standard operation procedure as a prerequisite for sound hazard assessment of nanomaterials.

Highlights

All over the world, different types of nanomaterials with a diversified spectrum of applications are designed and developed, especially in the field of nanomedicine
The collaborating laboratories involved in this intercomparison study were all part of the German DFG Priority Program SPP1313 “Biological Responses to Nanoscale Particles”[14] and involved laboratories at
In addition to the thorough NP characterization following the synthesis procedure, we carried out dynamic light scattering (DLS) measurements after incubating the NPs at 37 °C for 6, 12, 24, 48, and 72 h with Dulbecco’s Modified Eagle Medium (DMEM), containing 10% fetal calf serum

Summary

Introduction

Different types of nanomaterials with a diversified spectrum of applications are designed and developed, especially in the field of nanomedicine. It is already known that different laboratories may have used the same assay and nanomaterial for testing in the same cell line, variations in the experimental procedures still lead to inconsistent results In view of this situation we have decided to perform a carefully controlled round-robin experiment involving a number of different laboratories in Germany. In this intercomparison experiment we studied quasi-monodisperse NPs with a diameter in a similar size range (TEM diameter: 55 ± 2 nm and 74 ± 11 nm, hydrodynamic diameter about 120 nm) but of different chemical composition i.e. silica and polystyrene NPs, two of the most frequently used nanomaterials. The collaborating laboratories involved in this intercomparison study were all part of the German DFG Priority Program SPP1313 “Biological Responses to Nanoscale Particles”[14] and involved laboratories at

Methods

Results

Discussion

Conclusion