Nisin-loaded poly-L-lactide nano-particles produced by CO2 anti-solvent precipitation for sustained antimicrobial activity

Nisin-loaded poly-L-lactide (PLA) nano-particles were fabricated by processing protein/polymer organic solutions by semicontinuous compressed CO2 anti-solvent precipitation. Preliminary solubility studies were carried out for an optimised selection of organic solvent mixtures leading to preparation of protein/polymer solutions. The particles were prepared by processing 50:50 dimethylsulfoxide/dichloromethane mixtures containing 1% polymer (w/v) and 5 or 20% nisin (nisin/polymer, w/w). Proper operative conditions (organic solution injection rate, precipitation temperature and gas pressure, CO2 flow rate, washing time, etc.) were set up to yield production reproducibility, high product recovery (over 70%) and high drug loading (over 95% of the recovered protein). Scanning electron microscopy demonstrated that spherical, smooth surfaced particles were produced. Light scattering showed that the particle size was in the range of 200-400 nm and the products were characterised by narrow polydispersity. In vitro release studies showed that the protein is slowly released throughout 1000 h. However, the release was slower as the salt concentration and the pH of the release buffer increased. Solubility investigations suggested that the observed differences in protein release rate out of nano-particles was attributable to the protein interaction with the polymer which was found to increase as the pH or the salt concentration increased in the release buffer. In vitro studies carried out by nano-particle incubation in medium containing Lactobacillus delbrueckeii showed that nisin was released in the active form and the antibacterial activity was maintained up to 45 days incubation. (C) 2004 Elsevier B.V. All rights reserved.