Development of a procedure for coupling the homing device glu-plasminogen to liposomes.

The aim of this study was to find a suitable way of coupling the homing-device glu-plasminogen to the outside of liposomes. The described procedure is based on the reaction of thiol-groups introduced in the protein with thiol-reactive groups of the liposome. Details on the thiolation of proteins with the reagent succinimidyl-S-acetylthioacetate (SATA) were studied for a model-protein, amylase. Increasing the incubation-ratio SATA: amylase resulted in a gradually growing number of introduced thiol-groups, until a maximum of about 5 mol SH per mol amylase was reached. The enzymatic activity of the derivatized protein was even higher than that of native amylase. The thiol-introduction was then applied to glu-plasminogen itself. After activation with SATA, the protein was incubated with liposomes containing the thiol-reactive anchor maleimido-4-(p-phenylbutyrate)-phosphatidylethanolamine (MPB-PE). Under the chosen conditions, incubation of 0.5-2.5 mg/ml protein with 6.0-7.5 mumol/ml phospholipid for 30-120 min resulted in coupling-ratios of 20 to 94 micrograms glu-plasminogen per mumol phospholipid. This corresponds with about 140 to 660 protein molecules per liposome. SATA-derivatization of glu-plasminogen brought about a loss of its enzymatic activity induced by streptokinase. This activity of liposomally coupled plasminogen was about 52 to 74% of the activity of native glu-plasminogen (depending on the coupling-ratio). Although this may seem a significant loss of activity, it was shown that the capacity of liposomal glu-plasminogen to bind to its target, fibrin, was not reduced but several fold higher under the used conditions than that of the free protein. Therefore, the described method for thiol-introduction is an effective way to thiolate amylase without loss of activity, and to bind the homing-device glu-plasminogen to liposomes without substantially interfering with its fibrin-binding/homing capacity.