Characterization and morphological analysis of a cholecystokinin derivative peptide-loaded poly(lactide-co-glycolide) microspheres prepared by a water-in-oil-in-water emulsion solvent evaporation method

The water soluble peptide, pBC 264, derived from cholecystokinin and composed of seven aminoacids was encapsulated in poly(lactide-co-glycolide) (PLG) microspheres prepared by a multiple emulsion [(W-1/O)W-2] solvent evaporation method. The encapsulation efficiency of pBC 264 was very low when the inner emulsion contained no stabilizing agent. However, the encapsulation rate was improved by the addition of ovalbumin (OVA) used as stabilizer of the inner emulsion. In addition, the presence of a pH gradient between the inner and the outer aqueous phases of the multiple emulsion led to an increase of the retention of the peptide within the microspheres. Nevertheless, the molecular weight of the polymer did not have any influence on the encapsulation efficiency of the peptide. All microspheres batches showed a mean diameter below 10 mu m. In vitro release studies were carried out in both phosphate (pH 7.4) and TRIS buffer (pH 8), with microspheres made from several copolymers differing in their molecular weight. Release kinetics were characterized by a dramatic burst effect corresponding to the release of the major part of the entrapped pBC 264. DSC analysis were able to show that there was no interaction between OVA and the polymer suggesting that the protein is not dissolved in the polymer network but mainly present at the interfaces of the inner aqueous phase/polymer and microsphere surface/outer aqueous phase, The OVA that is present on microspheres might create a porous structure that was observed by electron microscopy. These pores would be responsible for the fast release of pBC 264. These results allow better comprehension of the effect of pH gradient and the use of OVA as a stabilizer on the encapsulation and release pattern of PLG microsphere-loaded small peptides.