POTENTIAL LOW TOXICITY CROSSLINKER FOR PROTEIN-BASED NANOPARTICLES

Abstract

Crosslinking is an essential procedure for maintaining the integrity of protein-based nanoparticles, but the application of toxic crosslinkers is usually undesirable. In this study, a tyrosinase-aided crosslinking procedure was developed and compared to a conventional crosslinker (i.e. glutaraldehyde). Nanoparticles were firstly synthesized from sodium caseinate (SC) in both aqueous and alcoholic solvent systems. The particles were crosslinked by tyrosinase (alone or with added natural phenols) or glutaraldehyde and then examined for their integrity under simulated environmental stress, including pH variation and solvent evaporation. Under aqueous condition, SC nanoparticles were not crosslinked sufficiently by tyrosinase or phenols alone, despite the abundance of tyrosine residues in SC. Conversely, satisfying crosslinking was achieved by tyrosinase combined with two natural phenols (catechol or chlorogenic acid, both at 2.5 mol/mol protein), as evidenced by stable particle size and count rate under environmental stress. A higher dose of 7.5 mol/mol protein was required for glutaraldehyde to achieve a comparable efficacy. Upon introduction of alcohol, the efficacies for both glutaraldehyde and tyrosinase-phenol mixtures decreased, but glutaraldehyde required lower dose and exhibited more significant crosslinking for achieving same crosslinking efficiency. However, a considerable number of nanoparticles were detected by scanning electron microscopy with both crosslinkers. Overall, tyrosinase-aided oxidation is a competitive, low-toxicity approach for crosslinking protein nanoparticles.