Water-in-Water Emulsions for Obtaining Enzyme-Loaded Microgels and Encapsulated Emulsions

Encapsulation of enzymes into protective matrices is of interest in drug delivery or industrial processes, to control the release of the enzyme or protect it from harsh environments. We are studying the encapsulation of lactase (β-Gal) into drug delivery vehicles, templated by water-in-water (W/W) emulsions. W/W emulsions are colloidal dispersions made of two immiscible aqueous phases that are in thermodynamic equilibrium, in absence of both oil and surfactant. This makes them of interest for environment friendly processes, in which organic solvents are replaced by aqueous ones, and for designing biocompatible delivery vehicles. Moreover, gelled droplets in the micron range, microgels, can be obtained by gelling and crosslinking the dispersed phase of W/W emulsions. Two distinct W/W emulsion systems were selected to serve as templates: sodium carboxymethylcellulose (NaCMC) / bovine serum albumin (BSA) mixtures and gelatin/maltodextrin mixtures. In the first system, the NaCMC/BSA mixture, phase behavior of the polymer mixture was analyzed, showing that this system can form W/W emulsions under basic pH conditions (pH 11-13). Emulsions with droplets between 5-20 µm were obtained. Ca2+ crosslinked selectively NaCMC, while the trivalent ions Fe3+ and Al3+ crosslinked both polymers, thus also the entire emulsion. By dropping the emulsion into the trivalent crosslinker solutions, encapsulated emulsions could be obtained, which consist of BSA gel beads that contain encapsulated NaCMC emulsion droplets. Freeze-drying of those beads lead to particles with pores, whose size corresponded to that of the emulsion droplets. Bead size was minimized down to ~600 µm by electrospraying the emulsion into the ion solutions. These beads, composed of both polymers, BSA and NaCMC, remained stable when simulating pH conditions experienced during the passage from food to the stomach over to the intestine, making it an interesting delivery vehicle for oral delivery of active molecules. The challenges of immobilizing enzymes into this type of encapsulated emulsions have been studied and discussed. In the second system, the gelatin/maltodextrin aqueous mixtures, the aim was to obtain gelatin microgels, crosslinked with genipin, to serve as enzyme carriers. The phase behavior of gelatin/maltodextrin mixtures in water was analysed in details. Microgels were formed from the gelatin-in-maltodextrin emulsions by cooling and crosslinking the dispersed gelatin droplets with genipin. Particle sizes as small as 6 µm were reached. Those microgels had a slight swelling response at pH values different from their isoelectric point (pI≈5) and shrank at increasing ionic strength. Crosslinking increased their stability in simulated gastric pH and temperature conditions. Microgels could also be kept in a dry form, by freeze-drying the suspensions. Various incorporation methods of the enzyme β-Gal were tested, to achieve highest encapsulation yield and activity recovery. Higher crosslinking degrees increased encapsulation yields. These conditions lead however also to the highest activity loss, due to direct contact between genipin and the enzyme, which partly deactivated the enzyme. Considering the activity loss, the highest activity recovery, which corresponds to active enzyme remaining inside the microgels, was achieved at intermediate crosslinking degrees. The enzyme remained active over at least one month, however a challenge was leakage of the enzyme from the microgels, which occurred faster at lower crosslinking rates. Therefore, of interest is the fact that the enzyme remained active after a complete cycle of freeze-drying and rehydration of enzyme-loaded microgel particles. The enzyme-loaded crosslinked gelatin microgels were not able to preserve enzyme activity under simulated gastric fluid temperature and pH conditions. It was shown however that they have some protective effect on enzyme activity at pH 5.8 and 37 °C. These can be considered as preliminary results for their possible use in e.g. industrial production of lactose-hydrolyzed milk, which has similar pH and temperature conditions