W1. NPs’ synthesis and functionalization
The AuNPs were obtained in two shapes, spherical and nanorods, having different sizes, 25, 55, and 100 nm for spherical NPs, while 15 x 55 and 15 x 100 nm, for nanorods. The obtained NPs were coated with a negatively charged polymer shell (PMA coating) and used as a platform for particle decoration with glycopeptides (A2G2S2) through EDC/NHS coupling reaction. The characterisation of the physico-chemical properties of the AuNPs after each step of the surface modification indicated obtaining high-quality, monodisperse, colloidally stable, and well-characterized glycan-NPs.
W2: Optimization of developed glycol-NPs and extracellular studies.
Here, we studied the binding accessibility of glyco-NPs in serum-free and protein-rich media using Sambucus Nigra Lectin (SNA), which is a protein bind preferentially to sialic acid attached to terminal galactose in the α-2,6 linkage of A2G2S2. The data indicated a clear binding of SNA to glyco-NPs, which confirmed its biological accessibility. The colloidal stability of the NPs (i.e. on their agglomeration behaviors) was studied in different physiological conditions (cell media, and different pH) using UV/Vis spectroscopy and DLS. Data showed that all the AuNPs samples exhibited high colloidal stability in complete cell media (DMEM, 10% FBS, 1% P/S) for at least 24 h and at a wide range of pH values (pH= 5 - 12).
W3: Protein corona evaluation and intracellular studies
To study the protein corona, the obtained NPs were exposed to increasing concentrations of blood plasma and Fetal bovine serum in order to mimic in vitro / in vivo conditions and, following incubation at 37°C, the corona-NP complexes were isolated and characterized by different techniques, including UV-vis spectroscopy, DLS, DCS, and SDS-PAGE. Overall, the SDS-PAGE showed comparable protein patterns, indicating that the surface modification had a low influence on the corona formation. However, the data obtained from DLS and DCS (differences in the hydrodynamic size of NPs and their relative apparent particle diameter) suggests that the glycan-coated NPs might reduce the corona formation compared to the PMA-NPs. Toxicity and cellular uptake studies were performed using hepatocyte (HepG2) and Raw264.7 cell lines. The data from the LDH assay showed no acute toxicity features after 24 h of exposure to different concentrations of AuNPs. Cellular uptake studies by flow cytometry and ICP-MS indicated less uptake of glycan-conjugated NPs by both types of cells compared to PMA-NPs, which indicates the ability of the glycan-coated NPs to escape from macrophage recognition.
Dissemination
•The results of this project were disseminated to the scientific community in the form of a poster and oral presentation at two international conferences.
•The results were communicated to the public society through a blog post at the European research night, at TCD (Dublin/ Ireland) on 30 Sep 2022.
•Jennifer Fernandez Alarcon*, Mahmoud Soliman*, Tanja Ludtke, Eva Clemente, Marko Dobricic, Martina B. Violatto, Alessandro Corbelli, Fabio Fiordaliso1, Chiara Cordiglieri, Laura Talamini, Giovanni Sitia, Sergio Moya, Paolo Bigini and Marco P. Monopoli, Long-term retention of gold nanoparticles in the liver is not affected by their physicochemical characteristics. The manuscript is submitted to the Journal of Nanobiotechnology.
Exploitation
The obtained results will provide (upon publication) a solid reference to scientific research groups working on synthesizing NPs with enhanced physical-chemical properties, and scientists in nanomedicine-related disciplines such as targeting, drug delivery & toxicology. It will also push all other nanomaterials to a novel strategy developed during this fellowship toward the progress of clinical translation of nanomedicine. The raw data obtained during this project will be also available via a public repository e.g. OpenAIRE, having first taken care to protect IP and any sensitive information.
