# Gold Nanoparticles and Nanorods in Nuclear Medicine: A Mini Review
## 3 In Vitro Evaluation of Radionuclides Delivered by AuNPs and AuNRs  
Studies in vitro are very important, because they improve knowledge of what happens at the molecular level when nanoparticles interact with biological systems (i.e., bloodstream, culture medium, and/or cell cytoplasm). Exploring these interactions is essential to understand the next biological processes such as distribution, metabolism, and elimination, etc., in vivo studies. Moreover, the in vitro evaluation of AuNPs and AuNRs presents different advantages such as lower cost, faster speed, and a lack of ethical concerns. The in vitro studies evaluate different properties relative to nanoparticles, such as the toxicity, the uptake into the cells, their stability in biological solutions in relation to size and shape, and particle-coating dose [117; 118]. In particular, the attachment of radionuclides could influence the surface properties and modify the capability of loading other targeting or therapeutic compounds. In the U87 MG glioma cell line, the targeting capability of the arginine-glycine-aspartic acid (RGD) peptide after \({}^{64}\)Cu integration in AuNRs was assessed. The RGD maintains its capability of targeting integrin after \({}^{64}\)Cu treatment [41]. Similarly, the attachment of PEG-trastuzumab (HER2-specific monoclonal antibody molecules) on the AuNPs surface (\(\mathcal{O}=5\) nm) does not change the \({}^{211}\)At absorption [82]. By contrast, the surfactant stabilizer CTAB, which is essential for the synthesis of AuNRs, exhibits strong cytotoxicity in terms of apoptosis and autophagy, and damage mitochondria producing intracellular reactive oxygen species (ROS) [119]. Of all the aforementioned properties, toxicity is the most studied. It can be defined as beneficial when radio gold-based nanomaterials are used for therapeutic treatments, or as detrimental when they are used for diagnostic procedures. The main endpoints studied to assess the toxicity in vitro are: (i) proliferation (i.e., MTT cell viability assay, clonogenic assay), (ii) apoptosis (i.e., annexin-V assay,Comet assay, TUNEL assay), (iii) membrane integrity (i.e., uptake of Trypan Blue (TB) or Neutral Red (NR) dyes), (iv) oxidative assays (i.e., evaluation of the content of reactive oxygen species), and (v) DNA damage [120; 121]. Cytotoxicity at different concentrations of the \({}^{198}\)AuNPs with dimension ranged from 25 to 30 nm and encapsulated in arabinaxl (AX) was determined in HeLa cells by NR dyes. The results show their non-toxic effect because they do not act on the cell viability and morphology (see Figure 4a,b). Consequently, the \({}^{198}\)AuNPs encapsulated into AX could be employed for in vivo studies in the field of diagnostics [122]. The cytotoxicity of the \({}^{124}\)I-AuNPs embedded into dendritic cells (DCs) used for immunotherapy has been evaluated in terms of proliferation by MTT cell viability assay and in terms of apoptosis by annexin-V assay. The cell viability test has shown no significant differences between the labeled and unlabeled DCs in the range of concentrations studied (1.0 to 4.0 nM) over 48 h. Moreover, \({}^{124}\)I-AuNP uptake in the DCs did not altered their migration and their antigen uptake capacities. Overall, these results point toward the use of these AuNPs as platforms for nuclear and optical imaging applications [123]. Furthermore, the cytotoxicity in terms of viability of \({}^{198}\)AuNPs (\(\mathcal{O}\) = 3.7 \(\pm\) 1.0 nm) integrated within the Lactobacillus bacteria cells (LACT) has been studied. The study was performed by using MTT test on breast cancer (MCF-7) and on normal colon (CRLA1790) cells, which were incubated with radioactive or non-radioactive LACT-AuNP. The results have shown in both cell lines a decrease of cell viability after incubation with LACT-\({}^{198}\)AuNP with the MCF-7 cells more radiosensitive than the CRLA1790 cells. Moreover, after incubation with non-radioactive LACT-AuNP, no cytotoxicity was observed in both cell lines [124].