Neuroprotective effect of a new Insulin-Nanogel system

Nanogels (NGs) have a great potential in the development of “smart” nanocarriers for (bio)molecular drugs and contrast agent for bioimaging. They are formed by physically or chemically crosslinked polymer networks, characterized by a large and flexible surface available for multivalent bioconjugations. NGs can be produced with high yields and through-puts by pulsed electron-beam irradiation of dilute aqueous solutions of water-soluble biocompatible polymers. In this work, a carboxyl functionalized nanogel system (NG), generated by pulsed e-beam irradiation of a semi-dilute poly(N-vinyl pyrrolidone) (PVP) aqueous solution in the presence of acrylic acid, with an average diameter in the 60-70 nm range (PDI<0.3) was used as a substrate to generate chemically stable insulin-grafted PVP NGs. In particular, grafting was carried out using human insulin without (PVP-g-insulin) or with fluorescein isothiocyanate labeling (PVP-g-insulin-FITC). The hydrodynamic dimensions of NGs before and after grafting (“naked NGs” and “grafted NGs”) were investigated by Dynamic Light Scattering. The PVP-g-insulin-FITC system was used in order to both quantify the conjugation degree of insulin to the nanoparticles by UV-vis spectroscopy and to study NGs localization in cell cultures. Different conjugation degrees were obtained by varying the reaction conditions. Biocompatibility tests of naked and insulin-grafted NGs were performed on neuroblastoma LAN5 cells by MTS assay. Colocalization of PVP-g-insulin-FITC NGs with activated insulin receptor was detected by immunohistochemistry technique and microscopical observations. Finally, the biological effect of insulin-grafted NGs was verified by activation of Akt and FOXO3a, two molecules involved in insulin signaling.