Citation: Pourali, P.; Neuhöferová, E.; Dzmitruk, V.; Benson, V. Investigation of Protein Corona Formed around Biologically Produced Gold Nanoparticles. Materials 2022, 15, 4615. https:// doi.org/10.3390/ma15134615 Academic Editor: Montserrat Colilla Received: 23 May 2022 Accepted: 24 June 2022 Published: 30 June 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). materials Article Investigation of Protein Corona Formed around Biologically Produced Gold Nanoparticles Parastoo Pourali 1,† , Eva Neuhöferová 1,† , Volha Dzmitruk 2 and Veronika Benson 1, * 1 Institute of Microbiology, Czech Academy of Sciences, 142 20 Prague, Czech Republic; parastoo.pourali@biomed.cas.cz (P.P.); neuhoferova.eva@gmail.com (E.N.) 2 Center of Molecular Structure, Institute of Biotechnology, Czech Academy of Sciences, 252 50 Prague, Czech Republic; volha.dzmitruk@ibt.cas.cz * Correspondence: benson@biomed.cas.cz † These authors contributed equally to this work. Abstract: Although there are several research articles on the detection and characterization of protein corona on the surface of various nanoparticles, there are no detailed studies on the formation, detec- tion, and characterization of protein corona on the surface of biologically produced gold nanoparticles (AuNPs). AuNPs were prepared from Fusarium oxysporum at two different temperatures and charac- terized by spectrophotometry, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and energy-dispersive X-ray spectroscopy (EDS). The zeta potential of AuNPs was determined using a Zetasizer. AuNPs were incubated with 3 different concentrations of mouse plasma, and the hard protein corona was detected first by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then by electrospray liquid chromatography–mass spectrometry (LC-MS). The profiles were compared to AuNPs alone that served as control. The results showed that round and oval AuNPs with sizes below 50 nm were produced at both temperatures. The AuNPs were stable after the formation of the protein corona and had sizes larger than 86 nm, and their zeta potential remained negative. We found that capping agents in the control samples contained small peptides/amino acids but almost no protein(s). After hard protein corona formation, we identified plasma proteins present on the surface of AuNPs. The identified plasma proteins may contribute to the AuNPs being shielded from phagocytizing immune cells, which makes the AuNPs a promising candidate for in vivo drug delivery. The protein corona on the surface of biologically produced AuNPs differed depending on the capping agents of the individual AuNP samples and the plasma concentration. Keywords: biologically produced gold nanoparticles; hard protein corona; capping agent; Fusarium oxysporum 1. Introduction Nanoparticles have special properties due to their larger surface area compared to their bulk material [1]. Due to their small size, they can easily interact with their environment and enter the body through different routes, such as inhalation, absorption through the skin, or ingestion [2]. Sometimes, some of them are injected into the body for biomedical purposes [2]. When they enter the human body unintentionally or intentionally, they are surrounded by bodily fluids immediately after internalization [2]. The surface of the nanoparticles is then covered by various types of macromolecules called “soft corona”, which is mostly composed of proteins. Over time, the soft corona is displaced by the “hard corona”, which has a higher binding affinity [3–5]. It has been widely reported that the type of macromolecules surrounding the nanoparticles, as well as the type and nature of the nanoparticles, such as surface charge, size, shape, solubility, and other physicochemical properties, are important for the composition of the corona [6,7]. Materials 2022, 15, 4615. https://doi.org/10.3390/ma15134615 https://www.mdpi.com/journal/materials