Trapping drug efficiency in liposomes produced by extrusion of freeze-thaw multilamellar vesicles.

treatment was employed. Phospholipids were purchased from Avanti Polar Lipids. The vcsiclcs (at a phospholipid concentration of 1 mg/ml in 30 mM-Tris/HCI buffer, pH 7.0, containing 0 . I M-NaCI) were prepared by sonication for 30 rnin in a water-bath sonifier at tcmperaturcs above the phasc transition of the corresponding phospholipid. The concentration of a-sarcin was determined by absorbance measurements and considcring its extinction coefficient at 280 nm 141. *-Sarcin-phospholipid complexes wcre prepared by adding the protein to freshly prepared vesicles and incubating at 37°C for 60 min. The proteolytic treatment was performed using N-tosyl-1.-phenylalanine chloromethyl ketone-treated trypsin at an enzymc/substrate weight ratio of I :SO. Aliquots were taken at different hydrolysis times, reduced with 3% ( v / v ) 2-mercaptoethanol and analyscd by 0.1% ( w / v ) SDS/polyacrylarnide-slab-gel electrophoresis [4'% (w/v) and IS'%, (w/v) acrylamide for the stacking and running gels. respectively]. The absorbance scans at 550 nm (on a Beckman DU-8 spectrophotometer) of the vacuumdried gels (staincd with Coomassic Blue) were used t o calculate the remaining intact protein after the trypsin treatment. The results obtained are given in Fig. 1. The protein was completely hydrolysed after 24 h o f trypsin treatment at 37°C. However. phospholipid vcsiclcs provided an cffcctivc protection for a-sarcin against proteolysis. The percentage of hydrolysis reached a plateau at about 30% protein hydrolysis after about 10 h o f trypsin treatment. Thus, about 70% of the a-sarcin molecules in saturated protein-phospholipid complexes were protected against . proteolysis. The total number o f lysine plus arginine residues in a-sarcin reprcsents about 15% of the total amino acid residues, which cnhanccs the importance of the protection o f the whole polypeptide chain against proteolysis caused by the lipid vesicles. The described interaction between a-sarcin and phospholipid vesicles may be involved directly in the molecular mechanism by which the protein enters tumour cell membranes.