3.2.1. AuNPs

The use of novel nanomaterials necessitates a better understanding of potential adverse e ffects on biological entities [49]. These e ffects are strictly dependent on physico-chemical properties of NPs and evaluation of cytotoxicity is critical, especially from a medical field application perspective [50]. A lot of studies were conducted to assess biocompatibility in order to ensure safe drug release with low toxicity. In general, high degree of biocompatibility is verified when NPs interact with the living organisms without triggering unacceptable toxic, thrombogenic, carcinogenic, and immunogenic responses [51].

The unique physico-chemical properties of AuNPs make them a powerful tool in the biomedical field. They are widely used in photothermal treatment of cancer and in drug and gene delivery. For their synthesis, auric salts such as HAuCl4 are used as starting materials to obtain stable AuNPs with a size ranging from 1–100 nm (Brust or Turkevish [52,53]). In recent years, green syntheses of AuNPs has also been developed to reduce solvents and reduce/cap agents' toxicity so that AuNPs can be synthetized from plant extracts or biomass [54,55]. AuNPs are easy to functionalize with a wide range of ligands ranging to polymer, DNA, peptides, RNA, and fluorescent molecules particularly suitable for bio-imaging applications [56]. Furthermore, various cytotoxicity studies demonstrated their low toxicity due to the inert nature of Au [57,58].

Regarding the biodistribution, a recent work demonstrated a rapid elimination of colloidal AuNPs from blood circulation in a rats model, and liver and spleen accumulation without inducing adverse effects or inflammation [59]. Au comprised in passion fruit-like nanoarchitectures (NAs) were studied in murine models demonstrating the excretion by renal and biliary pathways without nephrotoxicity up to 150 mg kg−<sup>1</sup> [60]. Some studies reported pro-inflammatory cytokines expressions induced by AuNPs in different organs of rats [61] and mice [62]. However, the cytotoxic effects were temporary and normalized in a short time. The biocompatibility of Au is strictly correlated to their size, shape, concentration, as well as surface area as demonstrated by Biswas's group [63]. They investigated the effects of Au spheres (5, 10, 20, 30, 50, and 100 nm), cubes (50 nm), and rods (10 × 90 nm) in cells in adult retinal pigment epithelial cell line 19 (ARPE-19). The cubes with an average diameter of 50 nm and spheres (50–100 nm) resulted in being biocompatible up to 750 μg of concentration. NPs with a smaller size (<30 nm) and nanorods (10 nm) showed higher toxicity, but they are more able to be uptaked by cells. Kim et al. [64] used C57BL/6 mice to demonstrate the ability of AuNPs (20 nm) to cross BRB and enriched retinal layers after intravenous administration. NPs greatly accumulated in neurons (75 ± 5%), followed by endothelial cells (17 ± 6%) and peri-endothelial glial cells (8 ± 3%) without affecting both viability of endothelial cells, astrocytes, retinoblastoma cells, and expression of functional biomolecules. After five weeks of post administration, the thickness of retinal layers was undisturbed without any damage. Bakri et al. [65] used Dutch-belted rabbits to test biocompatibility of AuNPs. They used two groups of rabbits receiving a 67 μmol/0.1 mL and 670 μmol/0.1 mL of AuNPs into one eye respectively by intravitreal injection. After one month, evident inflammation or adverse effects were not detected also at a high dosage. Soderstjerna et al. [66] used AuNPs and AgNPs with 19 and 89 nm citrate capped to test biocompatibility in in vitro cultured retinas using 800 NPs/cell. The retina tissue model offered a sustained long-term retinal organization and controlled serum-free conditions. This study demonstrated that low concentrations of Au and AgNPs have adverse effects on retina and can induce apoptosis and oxidative stress. A strong connection between NPs hydrodynamic diameter, surface functionalization/concentration and the biological effects is evident. In particular, the authors showed that NPs size should be large enough to avoid their fast flow into circulation, but small enough to be administrated and easy cross the BRB [67].

Starting from this assumption, the application of AuNPs is influenced by these parameters that should be taken in consideration to customize nanotools for a specific biomedical application.
