Investigating the biological activity of proteins immobilized on colloidal nanoparticles

Currently, nanoparticles (NPs) play an increasing role in biomedical research and clinical applications, thanks to their peculiar optical, physical and chemical properties. A great challenge in nanodiagnostics is the development of new nano-sized devices aimed to optimize the detection of primary cancer cells and metastases. The design of ideal nanoconjugates, containing bioactive ligands specific for targeting cancer cell, requires optimization of fundamental parameters involved in conjugation reactions: both functional conformation and proper orientation must be preserved. This characteristic determines bioactivity, avidity and targeting efficiency of the functionalized NPs. In the context of this thesis, different conjugation strategies were analyzed, focusing on the improvement of the biological activity of the immobilized protein. First of all, trastuzumab-functionalized pegylated iron oxide nanoparticles were synthetized and protein conformation analyzed using FTIR spectroscopy. This technique provides direct evidence of the extent of native structure preservation of the immobilized protein, in dependence of the conjugation strategy. Moreover, the possibility to control the ligand/peptide orientation on the nanoparticle surface is a fundamental step to optimize receptor recognition. An elegant strategy involves the use of fusion proteins containing a small enzyme (defined “capture protein”) capable of irreversibly cross-coupling with a suicide inhibitor anchored to the solid surface. Three different approaches have been analyzed: SNAP (O6-alkylguanine-DNA-transferase), HALO (haloalkane dehalogenase) and cutinase enzymes fused with specific proteins or small peptides for the selective targeting of breast cancer cells. Although targeted therapy with monoclonal antibody, or small portion of these proteins, is a major treatment currently employed in many cancers, the use of short peptides as targeting moieties of tumor receptors have several advantages. The possibility to exploit gold nanoparticles (AuNPs) properties, to form a self-assembled monolayer on AuNPs surfaces, allows to increase ligand-receptor target affinity/recognition. The capability of all these bioconjugation methods to specifically and selectively target breast cancer cells, was confirmed by flow cytometry (FACS), confocal laser scanning microscopy and transmission electron microscopy (TEM).