TRACKING METALLIC NANOPARTICLES FOR CANCER THERAPY: STRATEGIES FOR LABELLING.

Nanotechnology offers a promising opportunity for advancing cancer diagnosis and therapy, particularly using metallic nanoparticles. The nano-systems investigated in this work were iron based nanoparticles and gold nanorods, each exhibiting properties of significant biomedical relevance. Iron based nanoparticles can generate localized heating under alternating magnetic fields, making them suitable for magnetic hyperthermia. Gold nanorods display near-infrared surface plasmon resonance, enabling deep-tissue optical activation and efficient photothermal conversion. These two metallic carriers are moieties of multicomponent nanosystems for drug delivery and remote activated therapy. The aim of this project was to develop effective strategies to label these metallic nanoparticles for both in vitro and vivo tracking. To this end, a fluorophore with excitation/emission in the red band, was employed as fluorophore and conjugated to polyethylene glycol (PEG) spacers of different molecular weights. PEG was used to reduce fluorescence quenching by modulating the fluorophore–nanoparticle distance, and specific anchoring moieties were introduced to PEG chains for ensuring selective binding: an anionic-terminating linker (R1) for iron based nanoparticles and a nucleophile-terminating linker (R2) for gold nanorods. Iron based nanoparticles were decorated with fluorophore-PEG-R1 (low molecular weight PEG), which adsorbs onto the nanoparticle surface through a dynamic equilibrium process. The labelling was carried out using increasing molar feed ratios of fluorophore-PEG-R1/ iron based nanoparticles to determine the maximum grafting density. Saturation was reached at a 1000:1 fluorophore-PEG-R1/ iron based nanoparticles ratio, corresponding to a decoration efficiency of approximately 50% chains, and to a surface density of 0.45 chains/nm². DLS, fluorescence spectroscopy, and TEM analyses confirmed PEG corona formation, along with proportional increases in fluorescence intensity and hydrodynamic size up to the saturation point. Despite the use of the PEG as spacer for fluorophore / iron based nanoparticles distancing, the nanoparticles still induced a slight decrease of fluorescence. Gold nanorods were labelled with fluorophore-PEGn-R2 conjugate obtained with three molecular weights PEGs (low, middle, high) across molar feed ratios up to 2000:1 fluorophore-PEGn-R2/gold nanorods. In this system, the fluorescent PEG conjugates bind to the gold nanorods surface. Surface saturation was reached at higher feed ratios for the lower molecular weight fluorophore-PEG-R2 corresponding to maximum surface densities of approximately 1.0, 0.4, and 0.3 chains/nm² for the low, middle and high PEG length, respectively. Structural characterization revealed PEG-density increases in fluorescence intensity, hydrodynamic size, and ζ-potential. Fluorescence measurements further showed a progressive reduction in quenching with increasing PEG length, confirming that fluorophore/gold nanorods distancing plays a key role in limiting energy transfer to the nanoparticle core. Iron based nanoparticles and gold nanorods cell-association studies validate the fluorescent labelling strategy for nanoparticle in vitro/vivo tracking and demonstrated that decorated nanoparticle interaction with cell depends on the fluorophore-PEG conjugate surface density and PEG molecular weight. Confocal microscopy further confirmed gold nanorods cell association and perinuclear localization of decorated gold nanorods.