Encapsulation of Bowman-Birk soybean proteinase inhibitor within zwitterionic phospholipid multilamellar vesicles

In the present document we describe the possibility for encapsulating a water-soluble protein lacking α-helices into multilayer liposomes made of soybean zwitterionic phospholipid mixtures (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)) in order to prepare proteoliposomes. The influence of the PC/PE w/w ratio on the incorporation efficiency of the Bowman-Birk soybean proteinase inhibitor (BBI) into liposomes was studied. Increase in ionic strength did not inhibit the proteoliposome formation process. BBI encapsulation did not affect liposome sizes, surface charge and structural phospholipid organization. Confocal laser scanning microscopy showed that BBI was located in the central part of the spherical particle and between bilayers. The fluorescence intensity after anthracene-labelled phospholipids interaction with Rhodamin B-labelled BBI confirmed that the protein is deeply immersed into liposomes. The biological activity (antitrypsin and antichymotrypsin) estimation of the protein entrapped in liposomes showed that the protein active centers are spatially shielded. The effect of an ionic detergent on the activity of the encapsulated BBI confirms this hypothesis and suggests that this shielding is reversible. The proteoliposomes prepared seem to be promising formulations for BBI delivery.