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The diagnostic and therapeutic application of nanoparticles requires comprehensive knowledge of their interaction with the biomolecular surroundings. The formation of the protein corona on nanoparticles that were internalized by living cells is yet to be understood. In this study, we present a robust approach for the electrophoretic and mass spectrometric analysis of the hard protein corona composition formed in living cells on ~30 nm citrate-stabilized gold nanoparticles, i.e., the proteins with the highest affinity towards the gold nanoparticle surface. The gold nanoparticles were internalized by MCF-7 cells for 24 h followed by the extraction of the hard protein corona‑gold nanoparticle bioconjugates from living cell cultures. The extracted proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by ESI-Q-TOF-MS, which allowed to identify 108 hard corona proteins. The experiments were repeated with J774 macrophage cells with incubation times of 1.5 h, 3 h, 6 h, and 24 h, and the results showed that the hard protein corona remained unchanged over time. Therefore, the proposed experimental approach proved to be a valuable tool for identifying hard corona proteins of nanoparticles internalized by living cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Due to the increased use of engineered nanoparticles (NPs), their tracing in environmental and biological systems is of utmost importance. Besides their accumulation within a biological specimen, little is known about their degradation and transformation into corresponding low-molecular species that might influence any toxicological impact., Analytical Methods: Wistar rats underwent intraperitoneal injections of 40 nm citrate-stabilized gold nanoparticles. Different liver samples were analysed for the occurrence of nanoparticles and potential degradation products by means of spICP-MS, TEM and HPLC-ICP-MS., Main Findings: Studies using spICP-MS revealed the presence of the originally administrated Au NPs (40 nm diameter) and some evidences of other Au-containing species due to the increased background signal. Images obtained by transmission electron microscopy (TEM) showed the predominant presence of particles of significantly smaller diameter (6 ± 2 nm). As complementary method, HPLC-ICP-MS confirmed the presence of both particle types indicating a degradation of the Au NPs accompanied by detection of low-molecular Au species., Conclusions: This study underlines that degradation of gold nanoparticles to low-molecular gold species might have to be taken into account in future for studies on their toxicological behaviour and their potential use in clinical applications., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

The study of the uptake and distribution of elements in marine environments is of great interest for understanding their pathways and accumulation. Here, we investigated in laboratory experiments the accumulation behavior of Cd in the sea anemone Bunodosoma caissarum and the mussel Perna perna. Specimens were incubated with isotopically enriched 116 Cd in aquariums. Cd concentrations in the seawater and in the tissues of B. caissarum and P. perna were followed by inductively coupled plasma-mass spectrometry (ICP-MS) by means of isotope dilution analysis. Bioconcentration factors for B. caissarum and P. perna exposed to 0.9μg·L -1 of 116 Cd were determined to be 80.5 and 850, respectively. P. perna specimens exposed to 4.5μg·L -1 of 116 Cd reached 530. Cytosolic proteins associated with Cd from the tissues were extracted and further analyzed by size-exclusion chromatography coupled to ICP-MS. Cd accumulation could be detected in both organisms ranging from high-molecular to low-molecular species., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Once nanoparticles enter a biological system, it is known that their surface is instantly covered by the biomolecules present with preference to proteins. This protein corona has been a subject of numerous studies in order to reveal its composition. Besides that, growing interest exists in its quantitative determination in order to gain a deeper insight into the nature of these nanoparticle-protein bioconjugates. Only a few analytical methods are available nowadays, so the aim of this study is to provide a reliable and alternative methodology for the quantification of the protein corona. The suggested approach is based on the assumption that the total protein content within the corona can be correlated to its sulfur concentration due to the presence of cysteine and methionine as sulfur-containing amino acids. Once the most abundant proteins had been identified with the use of gel electrophoresis with subsequent peptide analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS), the isolated nanoparticle-protein conjugates were subjected to total analysis of sulfur and the corresponding metal being present in the nanoparticles by inductively coupled plasma-mass spectrometry (ICP-MS). The concept is exemplarily demonstrated on citrate-stabilized gold nanoparticles (GNPs) incubated with human serum. Two different purification procedures were tested in order to isolate the sought bioconjugates. 26 most abundant proteins could be identified and an average of approximately 40 S atoms per protein was calculated and used for further studies. ICP-MS analyses of S/Au ratios served for the quantification of the protein corona revealing an absolute number of proteins bound to the incubated GNPs. Two main results could be obtained for this specific system under the chosen experimental conditions: the number of proteins per GNP decreased with their size from 10 nm to 60 nm and the obtained values suggested that the protein corona in this specific case was theoretically formed either as a monolayer (60 nm GNPs) or as a multilayer (5-7 protein layers per 10 nm GNP). Studies with bovine serum albumin (BSA) as the model protein showed similar results.

Nanoclusters of noble metals like Ag and Au have attracted great attention as they form a missing link between isolated metal atoms and nanoparticles. Their particular properties like luminescence in the visible range and nontoxicity make them attractive for bioimaging and biolabelling purposes, especially with use of proteins as stabilising agents. In this context, this study intends the synthesis of a specific Au nanocluster covered by bovine serum albumin (BSA). It is shown that size-exclusion chromatography is feasible for the purification and isolation of the nanocluster. A mass spectrometric characterisation, preferably by ESI-MS, indicates the presence of an Au9@BSA nanocluster. Enzymatic digestion of the nanocluster with trypsin results in a significant increase of the fluorescence intensity at 650 and 710 nm, whereas complementary MALDI-MS studies are presented for the identification of generated peptides and show a distinctive pattern in comparison to the pure protein. It can be concluded that Au9@BSA might be, in future, an interesting candidate for in vitro studies of protease activities.