Enzyme nanovehicles: Histaminase and catalase delivered in nanoparticulate chitosan

Widespread skin allergies are of high societal concern. This pathology usually includes "histamine intolerance" and inflammatory processes affecting the skin. Oxidative stress, due to both intrinsic and environmental factors, leads to skin disorders, disease, and aging. Since the stratum corneum (SC) allows only small, lipophilic molecules to be absorbed through the skin, proteins, which are large amphoteric macromolecules, display limited bioavailability. The present study investigates the potential of chitosan nanoparticles as vehicle for two enzymes (catalytic proteins): catalase (CAT) and diamine oxidase (histaminase, DAO). Chitosan is an inexpensive, biocompatible, biodegradable, mucoadhesive, antibacterial, and antifungal biopolymer. Chitosan nanoparticles (CNP) have a high surface to volume ratio, hence high surface charge density and stronger biological activity than chitosan itself. CNP encapsulating DAO/CAT were prepared by using chitosans from different sources (shrimp and fungal). Nanoparticles were prepared by ionic complexation with sodium tripolyphosphate (TPP). Two different protein concentrations (0.5 mg/mL and 1.0 mg/mL) were investigated in the preparation of the nanoparticles. The resulting CNP were characterized by size (from 200 to 300 nm) and zeta potential (up to 9 mV) measurements, encapsulation efficiency (EE, up to 48%), loading capacity (LC, up to 42%), ratio of residual amino groups (RRAG, up to 14%), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and residual enzymatic activity. The antioxidant activity of the CNP encapsulating CAT was evaluated as well. The kinetics of protein release from the nanocomposites was monitored spectrophotometrically (for 160 h). Here we report that CAT was encapsulated with higher EE and residual enzymatic activity, while encapsulated DAO displayed better release profiles. Following encapsulation and release from CNP, both enzymes retained activity. The activities of DAO/CAT remained constant after five months in storage at -20 °C. These findings show that tailored nanosized chitosan affords enhancement to enzymes with low activity (like DAO) and shows promise as a carrier for therapeutic enzymes. Further investigations into its capability to increase their bioavailability are warranted.