One primary drawback of enzyme catalysis at industrial scale is the short-term service life of the enzymes, they lose their activity due to oxidation or other processes which results in less stability and a shorter lifetime thereby rendering them less efficient. An effective way to increase the stability of the enzymes is to attach them to nanoparticles. In this work, the polymer Eudragit^® L 100-55 sensitive to pH was used to prepare laccase polymeric nanoparticles by the double-emulsion solvent evaporation approach. The size and morphology of the nanoparticles obtained were evaluated—as well as the encapsulation efficiency and zeta potential. pH effect on activity and stability was compared between free and immobilized laccase. Their stability was also determined in a sequential assay involving acidic pHs up to alkaline ones. The nanoparticles had a spherical shape with a mean size of 147 nm, zeta potential of −22.7 mV at pH 7.0 and load efficiency of 87%. The optimum pH of both free and immobilized laccases was 3.0, being the nanoparticles more stable at acidic pHs. Thus, this would be the first report of obtaining laccase nanoparticles with potential application in animal feed due to the stability conferred to enzymatic activity at pHs similar to those present in the gastrointestinal tract of rabbits, which would allow their potential use in animal feed.

The key to success of proteins as biopharmaceuticals is to have in place an efficient drug delivery system that allows in gaining access to their target sites. A novel procedure of preparing ovoalbumin-loaded Eudragit® S100 microparticles, based on combining salting out and double emulsion-evaporation steps was developed. The ratio of a water miscible solvent (acetone and isopropanol) to a non-water-miscible solvent (chloroform) and salt addition to aqueous phase external were shown to be the primary determinants of size, polydispersity index (PI) and encapsulation efficiency (EE). Once optimized, using an organic phase of 3:0.5:1.5 (acetone:isopropanol:chloroform, v/v/v), further control was exerted using modification of acetone diffusion by alterations in MgCl2 concentration. Diffusion control, using 75% w/w MgCl2 solution, produced microparticles with a mean size of 26.3 µm, a good PI of 0.36 and 56.5 ± 0.5% EE. Electron microscopy showed the particles to be smooth and spherical. Ovoalbumin release studies using different buffers demonstrated immediate release in the buffer at alkaline pH. Calorimetry studies suggested that ovoalbumin existed in the microparticle as a molecular dispersion. Thus, Eudragit® S100 microparticles have great potential as oral carriers for delivery of proteins to the intestines. Key words: Double emulsion/solvent evaporation, emulsification-diffusion, microparticles, protein encapsulation.

One primary drawback of enzyme catalysis at industrial scale is the short term service life of the enzymes. Enzymes lose their activity due to oxidation or other processes which results in less stability and a shorter lifetime thereby rendering them less efficient. An effective way to increase the stability, longevity and reusability of the enzymes is to attach them to nanoparticles by applying the double-emulsion technique. In this work the polymer Eudragit® L 100-55 sensitive to pH was used to prepare laccase polymeric nanoparticles by the double-emulsion solvent evaporation approach. The size and morphology of the nanoparticles obtained was evaluated by Scanning Electron Microscope and particle size distribution was assessed by Photon Correlation Spectroscopy. Encapsulation efficiency and zeta potential were calculated. The effect of pH on laccase activity and stability was compared between free laccase and the immobilized one. Their stability was also determined in a sequential assay involving acidic pHs up to alkaline ones. The nanoparticles had a spherical shape with a mean size of 289 nm, zeta potential of -22.7 mV at pH 7.0, and load efficiency of 87%. The optimum pH of both free and immobilized laccases was 3.0, being the nanoparticles more stable at acidic pHs (2.0-4.0). Howevwe, this last kept 80% of enzyme activity at pH 2.0 approx., after 24 h. The polymer Eudragit® L 100-55 also conferred them resistance towards the pHs usually found at the gastrointestinal tract. These results suggest the potential use of nanoparticles as adjuvants in animal feed, serving as carriers for oral laccase delivery.