Preparation of protein nanoparticle by dynamic aggregation and ionizing-induced crosslinking

Abstract

Globular proteins can be considered as nanotools useful to perform a variety of chemical reactions. These biological macromolecules have very complex and detailed topological characteristics responsible for their several functions, which can be used for technological applications.Albumin is one of the few commercially available proteins with potential use in the preparation of nanomaterials by the bottom-up strategy. Albumin nanoparticles (NPs) can be prepared by several methods, but the preservation of the structure of the native protein in the final structure has not yet received too much attention.Our research group has recently reported the use of ionizing radiation to obtain albumin NPs. The irradiation of an ethanol solution of bovine serum albumin (BSA) allows obtaining protein NPs in the size range of 20?40 nm. However, a plausible mechanism to explain the process of preparation of protein-based NPs has not yet been described. In this work, we performed a series of experiments to prepare protein based NPs in two independent steps: (i) dynamic aggregation of BSA by ethanol and (ii) radiation-induced cross-linking by gamma or electron beam irradiation. Dynamic light scattering (DLS), circular dichroism (CD), Fourier Transform Infrared Spectroscopy (FT-IR) and UV spectroscopy measurements provided additional information about the conformation of BSA. No spectroscopy signal changes of aromatic amino acids were detected by UV and a loss of 20% of the alpha helix secondary structure was determined by CD. Drug-carrier functions were studied by binding and releasing assays of Merocyanine 540. BSA-NPs showed a drug-carrier behavior similar to that of BSA. Finally, we evaluated the possibility to prepare protein NPs containing more than one protein using the same procedure. Bi-protein NPs were prepared from lysozyme and BSA. The bi-protein NP showed enzymatic activity of lysozyme, which confirms the functionality of the NP prepared by this novel method.