Protamine-induced Aggregation of Unilamellar Liposomes

Abstract

Separation of non-encapsulated drug from unilamellar liposomes is critical for accurate estimation of encapsulation as well as to recover the non-encapsulated drug. The most commonly used methods for this purpose are either tedious or expensive. In this study we investigated the separation of free drug by aggregation of unilamellar liposomes by the addition of protamine. Several phospholipids and cholesterol were used in the preparation of liposomes. Stearylamine was added to provide positive charge and phosphatidylserine or dicetylphosphate for negative charge. Multilamellar liposomes (MLVs) were prepared by thin-film hydration and were converted to large unilamellar liposomes (LUVs) by passing through a 0.2 μm polycarbonate membrane using an extruder. Particle size of liposome preparations was characterized. Protamine solution in phosphate-buffered saline (PBS) was added to LUV preparations, vortex mixed and equilibrated overnight at room temperature. Aggregates of protamine-liposome were separated by centrifugation. The negatively charged liposomes aggregated immediately after the addition of protamine, whereas neutral and positively charged liposomes required overnight incubation. Also, the amount of protamine required for aggregation was significantly higher for neutral and positively charged liposomes (40 mg mL−1) compared with negatively charged liposomes (5 mg mL-1). The results indicated no leakage of encapsulated drug due to aggregation. It can be concluded that neutral as well as charged liposomes can be separated by protamine aggregation to estimate encapsulation efficiency as well as to separate non-encapsulated drug from liposomes.