Analysis of complexes formed by small gold nanoparticles in low concentration in cell culture media.

Stefán B Gunnarsson,Fredrik Johansson,Katja Bernfur,Tommy Cedervall,Ulrica Englund-Johansson,Amitava Mukherjee

doi:10.1371/journal.pone.0218211

Abstract

New nanomaterials are constantly developed with applications in everything from cosmetics to high tech electronics. Assessing their biological impact has been done by analysis of their adsorbed protein corona, in vitro cell assays, and larger scale ecotoxicological studies. This has proved to be a huge challenge due to the wide range of available nanomaterials and their unpredictable behaviour in different environments. Furthermore, the enormous number of experimental variables make comparisons difficult. Concentration is one of these variables and can vary greatly depending on the aim of the study. When analysing the protein corona, concentrations are often higher than in cell assays. Using a combination of complementary techniques, we have characterised 20 nm gold nanoparticles in a concentration level commonly used in cell studies. We compare their behaviour in a commonly used, protein rich medium and one protein poor medium over 24 hours. Under these conditions, the NPs were stable in protein rich environment but underwent gradual aggregation in protein poor medium. We characterise the biomolecular corona in both media. In protein poor medium, we can describe the often overlooked aggregation. The aggregates’ morphology is confirmed by cryo-TEM. Finally, in the protein poor medium, by infrared spectroscopy, we have identified the amino acid arginine in the biomolecular corona which drives the aggregation.

Highlights

The unique properties of nanostructured materials have led to an ever increasing number of applications in various fields, e.g. electronics, cosmetics, heavy industry, and in pharmaceuticals [1,2,3]
The amino acid, carbohydrate, and salt concentrations are high in both cell culture medium (CCM)
After establishing the NP aggregation behavior in the absence of proteins, we examined the effect of a protein rich environment

Summary

Introduction

The unique properties of nanostructured materials have led to an ever increasing number of applications in various fields, e.g. electronics, cosmetics, heavy industry, and in pharmaceuticals [1,2,3]. Our study describes the behavior of 20 nm gold nanoparticles (Au NPs) at the very low particle concentration of 58 pM. This concentration is close to the lower detection limit of the applied analytical methods. We used two different CCM (expansion and RPMI) equilibrated in 5% CO2 mixed with Au NPs. The Au NPs were diluted 1:20 in the CCM, which gives final gold concentration of 2.8 μg/mL or 58 pM, and incubated in sterilized Eppendorf tubes until they were analyzed. Low concentrations of gold nanoparticles in cell media of Au NP stock solution supernatant after centrifugation was added, to account for any contribution of the citrate stock solution to the sample pH. Each sample was measured 50–100 times to increase signal to noise ratio

Results and discussion

Summary of methodological approach and results

Conclusions