CONJUGATION STRATEGIES OF INSULIN TO NANOGELS: A HOPE FOR ALZHEIMER'S DISEASE THERAPY?

The importance of insulin in ageing as well as its role in cognition and other aspects of normal brain functions are well established. Recently, a growing body of evidence links insulin resistance and insulin action to neurodegenerative diseases, especially Alzheimer's disease (AD), one of the most common form of dementia. Insulin, insulin receptor (IR) and IR signaling have been shown to be involved in brain cognitive functions and their dysfunction in aging brain degeneration. Thus, insulin administration could be a potential therapeutic agent for AD. To the purpose of crossing the blood-brain-barrier (BBB), nanotechnologies can be a valuable help. 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. Co-localization 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.