Effects of salt addition on the microencapsulation of proteins using W/O/W double emulsion technique

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Abstract
The influence of co-encapsulation of stabilizing additives together with BSA on microsphere characteristics using the modified water-in-oil-in-water emulsion solvent evaporation (W/O/W) method was investigated. For this purpose, poly(L-lactide) microspheres containing bovine serum albumin (BSA) were prepared. The morphology, porosity, specific surface area, particle size, encapsulation efficiency and kinetics of drug release of protein loaded microspheres were analysed in relation to the influence of co-encapsulated stabilizing additives such as electrolytes. High salt concentrations in the internal (W1) aqueous phase, often necessary to stabilize protein or antigen solutions, led to an increase in particle size, particle size distribution, porosity and specific surface area. Bulk density and encapsulation efficiency decreased. The release profile was characterized by a high initial burst due to the highly porous structure. Addition of salt to the external or continuous water phase (W2), however, stabilized the encapsulation process and, therefore, resulted in improved microsphere characteristics as a dense morphology, a reduced initial burst release, a drastically increased bulk density and encapsulation efficiency. Analysis of the specific surface area (BET) showed that the addition of salt to W2, regardless of the salt concentration in the W1 phase, decreased the surface area of the microspheres approximately 23-fold. Microsphere properties were influenced by salts additions through the osmotic pressure gradients between the two aqueous phases and the water flux during microsphere formation. Release profiles and encapsulation efficiencies correlated well with the porosity and the surface area of microspheres. Furthermore, the influence of a low molecular weight drug and different time-points of salt addition to W2 on microsphere characteristics were studied by encapsulation of acid orange 63 (AO63), confirming the results obtained with BSA. This study suggests that modification of the external water phase by adding salts is a simple and efficient method to encapsulate stabilized protein solution, with high encapsulation efficiency and good microsphere characteristics.

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