The Turkevich method was optimized to prepare gold nanoparticles (AuNP) stabilized by polyethyleneglycol (PEG) for µCT. Using various independent modalities, we thoroughly characterized the optimized PEG-AuNPs. Here, we show that PEG-AuNPs are retained in the blood and provide a high contrast in the high-resolution µCT imaging of blood vessels and inner organs. The biodistribution is characterized by prolonged circulation in the blood and accumulation in the liver, spleen and skin. The accumulation of AuNP in the skin resulted in the blue discoloration of eyes and the whole skin. In vitro experiments using a leukemic monocyte THP-1 cell line model expressing high levels of NLRP3 demonstrated that the NLRP3inflammasome was not activated by PEG AuNP. Over 9 months, the mice were scanned by µCT and were in good health. Scans in mice using PEG-stabilized AuNPs in this study were sharper, with a higher contrast, when compared to a commercial contrasting agent at the same dose. The PEG-AuNPs were morphologically and chemically stable for at least two years when stored in the refrigerator.

Department of Analytical Chemistry University of Chemistry and Technology Prague Technická 5 166 28 Praha 6 Czech Republic

Department of Histology and Embryology Faculty of Medicine Masaryk University Kamenice 753 5 62500 Brno Czech Republic

Department of Immunology Faculty of Medicine and Dentistry Palacky University Olomouc Hněvotínská 3 775 15 Olomouc Czech Republic

Institute of Clinical Immunology and Allergology University Hospital Hradec Kralove and Faculty of Medicine in Hradec Kralove Charles University Sokolská 581 50005 Hradec Kralove Czech Republic

Institute of Physics Czech Academy of Sciences Na Slovance 2 18200 Prague 8 Czech Republic

Institute of Scientific Instruments v v i AS CR Královopolská 147 612 00 Brno Czech Republic

Neurology Department The International Clinical Research Center ICRC of St Anne's University Hospital in Brno Pekařská 53 656 91 Brno Czech Republic

Veterinary Research Institute v v i Hudcova 296 70 621 00 Brno Czech Republic

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