Entrapment of proteins in phosphatidylcholine vesicles

Phospholipid vesicles have been used as vehicles to deliver macromolecules into cells. In this study, the entrapment of globular proteins of various molecular weights was investigated in four different types of phospholipid vesicles. These vesicles were generated in a mixture of ¹²⁵I-proteins of various molecular weight. The trapped protein is separated from untrapped by ultrafiltration and by two consecutive gel filtrations, Sepharose 4B and Sephadex G-100. The entrapped protein is analyzed by SDS-PAGE on slab gels and autoradiography. Entrapment of the proteins was demonstrated by their resistance to trypsin and chymotrypsin digestion. The relative amount of each entrapped protein was then compared to that of the original protein solution. In multilamellar vesicles and large unilamellar vesicles, proteins of molecular weights up to 97,000 showed identical trapping efficiency as sucrose. In small unilamellar vesicles generated by either sonication or ethanol injection method, however, the relative trapping efficiency of the protein decreased progressively as the molecular weight of the protein became greater. For example, the trapping efficiency of α-amylase (m.w. 97,000) was only half of that for sucrose. The apparent decrease in trapping efficiency with proteins molecular weight can be accounted for by the combination of bound water layer at the vesicle internal surface and the steric hindrance when protein is captured during vesicle formation.