Synthesis of papain nanoparticles by electron beam irradiation - A pathway for controlled enzyme crosslinking.

Crosslinked enzyme aggregates comprise more stable and highly concentrated enzymatic preparations of current biotechnological and biomedical relevance. This work reports the development of crosslinked nanosized papain aggregates using electron beam irradiation as an alternative route for controlled enzyme crosslinking. The nanoparticles were synthesized in phosphate buffer using various ethanol concentrations and electron beam irradiation doses. Particle size increase was monitored using dynamic light scattering. The crosslinking formation by means of bityrosine linkages were measured by fluorescence spectra and the enzymatic activity was monitored using Na-Benzoyl-dl-arginine p-nitroanilide hydrochloride as a substrate. The process led to crosslinked papain nanoparticles with controlled sizes ranging from 6 to 11nm depending upon the dose and ethanol concentration. The irradiation atmosphere played an important role in the final bioactivity of the nanoparticles, whereas argon and nitrous oxide saturated systems were more effective than at atmospheric conditions in terms of preserving papain enzymatic activity. Highlighted advantages of the technique include the lack of monomers and crosslinking agents, quick processing with reduced bioactivity changes, and the possibility to be performed inside the final package simultaneously with sterilization.

Radiation-synthesized protein-based drug carriers: Size-controlled BSA nanoparticles.

Nanotechnology has broadened the options for the delivery of agents of biotechnological and clinical relevance. Currently, attention has been driven towards the development of protein-based nanocarriers due to high the biocompatibility and site-specific delivery. In this work we report radiation-synthesized bovine serum albumin nanoparticles as an attempt to overcome limitations of available albumin particles, as a novel route for the development of crosslinked protein nanocarriers for the administration of chemotherapic agents or radiopharmaceuticals. Albumin containing phosphate buffer solutions were irradiated using γ-irradiation at distinct cosolvent concentrations-ethanol or methanol. Nanoparticle size was followed by DLS and bityrosine crosslinking formation using fluorescence measurements and SDS-PAGE. In addition, computational experiments were performed to elucidate the mechanism and pathways for the nanoparticle formation. The synthesis of BSA nanoparticles using γ-irradiation in the presence of a cosolvent allowed the formation of the nanoparticles from 7 to 70 nm without the use of any chemical crosslinker as confirmed by SDS-PAGE and DLS analysis. The combination of cosolvent and γ-irradiation allowed a fine tuning with regard to protein size.

Cyclodextrins: an overview of the complexation of pharmaceutical proteins.

Cyclodextrins are oligosaccharides, specifically cyclic alpha-1,4-D-glucose oligomers, that possess a cone-like shape resulting in a hydrophobic inner cavity capable of forming complexes with several guest molecules in a hydrophilic matrix. This capability has led to an extensive investigation into cyclodextrin applications in several different substrates with the purpose of overcoming limitations, such as solubility issues, physical degradation and sensitivity to solvents, in guest substances. Researchers have recently described successful interactions between cyclodextrins and proteins, such as enzymes, peptides and amino acids. These complex biomolecules consist of potent active ingredients and are employed in several industrial biocatalyst processes. However, this group in particular tends to have limited usage in pharmaceuticals due to its natural processes of degradation and instability in unusual environments, frequently requiring accurate procedures and stabilization methods in all stages of production. In several cases, the final product still has a short shelf-life and often requires a controlled environment for storage. The formation of a cyclodextrin supramolecular complex could not only prevent such problems, but also enhance the intrinsic characteristics of guest substances, thus allowing for an expansion in their industrial production and application. This work focuses on cyclodextrin interactions with protein-like structures in order to describe their possible applications in the formulation of pharmaceutical proteins.