Conjugation strategies of insulin to nanogels and biological activity tests

Nanogels (NGs) have a great potential in the development of "smart" nanocarriers for (bio)molecular drugs and diagnostics. They are formed by physically or chemically crosslinked polymer networks and 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, 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 labelling (PVP-g-Insulin-FITC). The hydrodynamic dimensions of NGs before and after grafting ("naked NGs" and "grafted NGs") were investigated by Dynamic Light Scattering. Average diameters were always in the 90-100 nm range, with narrow particle size distribution (PDI<0.3). The PVP-g-Insulin-FITC system was used in order to both quantify the conjugation degree of Insulin and evaluate its biological activity. Different conjugation degrees were obtained by varying the reaction conditions. Biocompatibility test 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 Foxo3, two molecules involved in Insulin signaling.