Possibility of encapsulation of water-soluble proteins into multilayer liposomes of soybean zwitterionic phospholipid mixtures (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)) was investigated. The influence of the PC/PE ratio (w/w) on efficiency of incorporation of the Bowman-Birk soybean proteinase inhibitor (BBI) and aprotinin (BPTI) into liposomes was studied. Protein encapsulation did not affect liposome sizes. Confocal laser scanning microscopy demonstrated that proteins were located in the central part of the spherical particle and also between bilayers. The study of biological (antitrypsin and antichymotrypsin) activity demonstrated partial spatial shielding of active sites of proteins entrapped in liposomes. The effect of an ionic detergent on the activity of the encapsulated BBI and BPTI is consistent with this hypothesis and suggests that this shielding is reversible. Stability of liposomes was examined using three various media modeling gastrointestinal fluids (gastric and intestinal juices and fluids). Data obtained indicate that the prepared liposomes seem to be promising formulations for BBI and BPTI delivery.

Lipids represent 20% of the total weight of the dried pool of medicinal leech salivary gland secretion (SGS) obtained from about 50 individual animals. SGS lacks phospholipids, but contains steroids. Immunochemiluminescent analysis of SGS revealed the presence of free steroid hormones: cortisol, progesterone, testosterone, estradiol, and dehydroepiandrosterone. Micro-chromatographic-mass spectrometric analysis of SGS and its low molecular weight fraction (LMW) (molecular masses ranged from 220 to 850 Da) has shown the multicomponent nature of the LMW fraction. Using standard preparations as the reference steroid hormones (cortisol, dehydroepiandrosterone, androstenedione, and testosterone) and histamine and serotonine have been identified in SGS.

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.

The interaction of native Bowman-Birk soybean protease inhibitor (BBI) and its hydrophobized derivative with multilamellar vesicles of various soybean phospholipids was investigated. Decrease in pH and introduction of negatively charged components to the lipid mixture increased BBI content in the protein-lipid complex. This suggests a contribution of electrostatic forces in the protein-lipid interaction. Protein hydrophobization insignificantly influenced BBI binding to lipids. In the complex with lipids, both proteins (BBI and its hydrophobized derivative) retained high anti-chymotrypsin activity (75–100%), which was not influenced by the presence of the ionic detergent sodium deoxycholate.