Production of liposomes using microengineered membrane and co-flow microfluidic device

Two modified ethanol injection methods have been used to produce Lipoid® E80 and POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes: (i) injection of the organic phase through a microengineered nickel membrane kept under controlled shear conditions and (ii) injection of the organic phase through a tapered-end glass capillary into co-flowing aqueous stream using coaxial assemblies of glass capillaries. The organic phase was composed of 20 mg ml−1 of phospholipids and 5 mg ml−1 of cholesterol dissolved in ethanol and the aqueous phase was ultra-pure water. Self-assembly of phospholipid molecules into multiple concentric bilayers via phospolipid bilayered fragments was initiated by interpenetration of the two miscible solvents. The mean vesicle size in the membrane method was 80 ± 3 nm and consistent across all of the devices (stirred cell, cross-flow module and oscillating membrane system), indicating that local or temporal variations of the shear stress on the membrane surface had no effect on the vesicle size, on the condition that a maximum shear stress was kept constant. The mean vesicle size in co-flow microfludic device decreased from 131 to 73 nm when the orifice diameter in the injection capillary was reduced from 209 to 42 μm at the aqueous and organic phase flow rate of 25 and 5.55 ml h−1, respectively. The vesicle size was significantly affected by the mixing efficiency, which was controlled by the orifice size and liquid flow rates. The smallest vesicle size was obtained under conditions that promote the highest mixing rate. Computational Fluid Dynamics (CFD) simulations were performed to study the mixing process in the vicinity of the orifice.