###### Abstract  
Gold nanoparticles (AuNPs) radiolabeled with therapeutic and diagnostic radioisotopes have been broadly studied as a promising platform for early diagnosis and treatment of many diseases including cancer. Our main goal for this study was the comparison of the biodistribution profiles of four different concentrations of gold nanocomposites radiolabeled with Technetium-99m (\({}^{99\text{m}}\)Tc). More specifically, AuNPs with an average diameter of 2 nm were functionalized with a tridentate thiol ligand. Four different concentrations were radiolabeled with \({}^{99\text{m}}\)Tc-tricarbonyls with high radiolabeling yields (>85%) and were further purified, leading to radiochemical purity of >95%. In vitro stability of the radiolabeled nanoconstructs was examined in cysteine and histidine solutions as well as in human serum, exhibiting robust radiolabeling up to 24 h post-preparation. Moreover, in vitro cytotoxicity studies were carried out in 4T1 murine mammary cancer cells. In vivo tracking of the radiolabeled nanoconjugates at both concentrations was examined in normal mice in order to examine the effect of AuNPs' concentration on their in vivo kinetics. Our work demonstrates that varying concentrations of radiolabeled AuNPs lead to notably different biodistribution profiles. technium-99m; gold nanoparticles; radiolabeling; cytotoxicity; biodistribution +
Footnote †: journal: Medical and Kapodistrian University of Athens, Mikras Asias 75, 11527 Athens, Greece  
Footnote †: journal: Medical and Kapodrian University of Athens, Mikras Asias 75, 11527 Athens, Greece
order to avoid spleen filtration and concluded that the maximum size was 150 nm [9]. [PERSON] et al. studied the biodistribution of AuNPs of a huge variety of sizes (15, 50, 100 and 200 nm) in mice. The results indicated that the smaller particles (15 and 50 nm) had higher blood concentration 24 h post-administration, while the 15 nm nanoparticles had a higher uptake in organs such as the lung, kidney and brain when compared to the larger-sized nanoparticles. An increasing trend in spleen uptake was again observed with an increase in particle size [10]. Furthermore, [PERSON] et al. demonstrated that smaller-sized nanoparticles clear more efficiently from the injection site (peritoneum) when compared to larger-sized microparticles in the same time frame [11]. Hence, nanoparticle size has a significant impact on their biodistribution. Nanoparticle size also influences therapeutic delivery aspects such as blood circulation half-life, cellular uptake and tumor penetration ability [4]. Surface charge is also important for the in vivo fate of AuNPs, with several studies revealing the impact in mouse models, since it affects the nanoparticle's blood circulation time and tumor uptake. [PERSON] et al. suggested the substantial role of surface charge on the in vivo behavior of nanoparticles, providing insights for improved therapeutic design. After intravenous injection, neutral and zwitterionic particles exhibit low clearance, while negative particles show rapid clearance due to moderate systemic exposure. After intraperitoneal injection, both positively and negatively charged particles are not sufficiently absorbed, indicating a difficulty in crossing the peritoneal barrier. The ability of surface charge to affect the in vivo behavior is crucial for developing nanotherapeutics for enhanced tumor uptake through both passive and active targeting [7]. Other studies have also shown that particles with positive charge tend to accumulate more in the kidneys, while negatively and uncharged particles showed higher uptake in the liver [8]. The route of administration also has an important impact on the in vivo kinetics of nanoparticles. [PERSON] et al. evaluated the differences between three methods of administration (intratumoral (i.t. ), intravenous (i.v.) and intraperitoneal (i.p.)) in order to identify the optimal administration route of a novel nanosystem of hexepeptides and AuNPs (P12) in healthy mice and mice with acute lung injury (ALI).