Effects of temperature, acyl chain length, and flow-rate ratio on liposome formation and size in a microfluidic hydrodynamic focusing device

Abstract

Microfluidic hydrodynamic focusing of an alcohol–lipid mixture into a narrow fluid stream by two oblique buffer streams provides a controlled and reproducible method of forming phospholipid bilayer vesicles (i.e., liposomes) with relatively monodisperse and specific size ranges. Previous work has established that liposome size can be controlled by changing the relative and absolute flow rates of the fluids. In other previous work, a kinetic (non-equilibrium) theoretical description of the detergent dilution liposome formation method was developed, in which planar lipid bilayer discs aggregate until they become sufficiently large to close into spherical liposomes. In this work, we show that an approximation of the kinetic theory can help explain liposome formation for our microfluidic method. This approximation predicts that the liposome radius should be approximately proportional to the ratio of the membrane bending elasticity modulus to the line tension of the hydrophobic edges of the lipid bilayer disc. In combination with very fast microfluidic mixing, this theory enables a new method to measure the ratio of the elasticity modulus to the line tension of membranes. The theory predicts that the temperature should change the liposome size primarily as a result of its effect on the ratio of the membrane bending elasticity modulus to the line tension, in contrast to previous work on microdroplet and microbubble formation, which showed that the effect of temperature on droplet/bubble size was primarily due to viscosity changes. In agreement with theory, most membrane compositions form larger liposomes close to or below the gel-to-liquid crystalline phase transition temperature, where the membrane elasticity modulus is much larger, and they have a much smaller dependence of size on temperature far above the transition temperature, where the membrane elasticity modulus is relatively constant. Other parameters modulated by the temperature (e.g., viscosity, free energy, and diffusion coefficients) appear to have little or no effect on liposome size, because they have counteracting effects on the lipid aggregation rate and the liposome closure time. Experiments are performed using phospholipids with varying hydrophobic acyl chain lengths that have phase transition temperatures ranging from −1 °C to 55 °C, so that the temperature dependence is examined below, above, and around the transition temperature. In addition, the effect of IPA stabilizing the edges of the bilayer discs can be examined by comparing the liposome sizes obtained at different flow-rate ratios. Finally, polydispersity is shown to increase as the median liposome size increases, regardless of whether the change in size is due to changing temperature or flow-rate ratio.