Synthesis and characterization of a new insulin conjugated nanogel for biomedical application

Insulin, a metabolic hormoneinvolved in glucose metabolism, plays also a neuroprotective rolein the central nervous system being able to revert the cytotoxic processes induced byA ? 42 ,a peptide involved in Alzheimer's disease. To reach the brain insulin have to across the BBB therefore an additional delivery strategy results to be necessary. For these aim we performed an insulin conjugated nanogels (NGs-In). 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 diameterinthe 60-70 nm range (PDI<0.3) was used as asubstrate to generate chemically stable insulin-grafted PVP NGs. In particular, grafting was carried out using human insulin without (PVP-ginsulin) 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. For the biological application, as first step, we have evaluated the biocompatibility and immunogenicity of NGs, at different concentration, on neuroblastoma LAN5 cells and PBMCs. Moreover we have demonstrated the capacity of NGs to protect the insulin from protease action by a resistance proteinase assay. Finally, the biological effect and neuroprotection of NGs-In has been verified