Ultrasmall gold nanoparticles functionalized with fluoresceinamine: a combined computational and experimental study of cellular uptake and fluorescence enhancement

Nanomaterials that enhance the fluorescence emission intensity of conventional fluorophores have broad applications, from cellular biology to real-time molecular bioimaging. This study combined computational and experimental approaches to evaluate the cytotoxicity and optical properties of hybrid gold nanoparticles (1.95 ± 0.55 nm) conjugated with fluoresceinamine (AuNPs–FLA). Computationally, interaction energies between FLA and Au clusters containing 1, 3, 5, or 7 atoms were calculated using density functional theory with the B3LYP functional and 6-311G** and LANL2DZ basis sets. The most stable complex involved using a 3-atom cluster interacting with the primary amine of FLA, consistent with the electron localization function indicating the N-Au interaction. Experimentally, UV–Vis, infrared, and fluorescence spectra of AuNPs–FLA and their precursors were compared. Additionally, in vitro cytotoxicity was assessed using the MTT assay in human foreskin fibroblasts (HFF), malignant glioblastoma multiforme cells (U251), and breast cancer cells (MCF-7). Both AuNPs and AuNPs–FLA showed minimal cytotoxicity at low and moderate concentrations (0.47–3.75 µg/mL), maintaining cell viability above 85%. Notably, AuNPs–FLA treatment resulted in an approximately four-fold increase in intracellular fluorescence intensity in HFF cells compared to free FLA (median 829 vs. 211; p < 0.0001), as determined by confocal microscopy, indicating enhanced fluorophore internalization upon Au conjugation. In addition, the AuNPs–FLA complex exhibited greater intracellular stability than free FLA for up to 1.5 h. These findings suggest that combining AuNPs with FLA holds promise for advanced imaging diagnostics and therapeutic applications in fields such as medicine, biotechnology, and biomedical research.