Cystatin incorporated in poly(lactide-co-glycolide) nanoparticles: development and fundamental studies on preservation of its activity

Preservation of biological activity is still a major challenge for successful formulation and delivery of protein drugs. Cystatin, a potential protein drug in cancer therapy, was incorporated in poly(lactide-co-glycolide) nanoparticles by the water-in-oil-in-water emulsion solvent diffusion technique. In order to preserve the biological activity of cystatin, a specific modification of the method of producing nanoparticles was introduced. The activity of cystatin was strongly influenced by the stirring rate during preparation and, to a lesser extent, by selected organic solvents. A synergistic effect of mechanical stirring and sonication, both at low energy levels, enabled nanoparticles to be formed without denaturing the cystatin. Nanoparticles produced by the optimised method ranged from 300 to 350 nm in diameter with 85% of the starting cystatin activity. The loading efficiency of cystatin depends on polymer type and ranged from 12 to 57%, representing an actual loading of 0.6-2.6% (w/w). Among various cryo-/lyoprotectants bovine serum albumin was identified as the most successful. The use of a protein protectant prior to nanoparticle formation was essential to maintaining the biologically active three-dimensional structure of cystatin. In addition, a specific type of poly(lactide-co-glycolide) polymer, particularly in terms of its functional groups, was identified to be important in retaining cystatin activity. Cystatin incorporated into nanoparticles in this way maintains its structural integrity, making it suitable for effective drug delivery. (C) 2004 Elsevier B.V. All rights reserved.