Traditional liposome preparation methods are based on mixing of bulk phases, leading to inhomogeneous chemical and/or mechanical conditions during formation; hence liposomes are often polydisperse in size and lamellarity. Here we show the formation of liposomes that encapsulate reagents in a continuous two-phase flow microfluidic network with precision control of size from 100 to 300 nm by manipulation of liquid flow rates. We demonstrate that by creating a solvent-aqueous interfacial region in a microfluidic format that is homogeneous and controllable on the length scale of a liposome, we can facilitate the fine control of liposome size and polydispersity.

We investigate the formation of unilamellar lipid vesicles (liposomes) with diameters of tens of nanometers by controlled microfluidic mixing and nanoparticle determination (COMMAND). Our study includes liposome synthesis experiments and numerical modeling of our microfluidic implementation of the batch solvent injection method. We consider microfluidic liposome formation from the perspective of fluid interfaces and convective-diffusive mixing, as we find that bulk fluid flow parameters including hydrodynamically focused alcohol stream width, final alcohol concentration, and shear stress do not primarily determine the vesicle formation process. Microfluidic device geometry in conjunction with hydrodynamic flow focusing strongly influences vesicle size distributions, providing a coarse method to control liposome size, while total flow rate allows fine-tuning the vesicle size in certain focusing regimes. Although microfluidic liposome synthesis is relatively simple to implement experimentally, numerical simulations of the mixing process reveal a complex system of fluid flow and mass transfer determining the formation of nonequilibrium vesicles. These results expand our understanding of the microfluidic environment that controls liposome self-assembly and yield several technological advances for the on-chip synthesis of nanoscale lipid vesicles.

A new method to tailor liposome size and size distribution in a microfluidic format is presented. Liposomes are spherical structures formed from lipid bilayers that are from tens of nanometers to several micrometers in diameter. Liposome size and size distribution are tailored for a particular application and are inherently important for in vivo applications such as drug delivery and transfection across nuclear membranes in gene therapy. Traditional laboratory methods for liposome preparation require postprocessing steps, such as sonication or membrane extrusion, to yield formulations of appropriate size. Here we describe a method to engineer liposomes of a particular size and size distribution by changing the flow conditions in a microfluidic channel, obviating the need for postprocessing. A stream of lipids dissolved in alcohol is hydrodynamically focused between two sheathed aqueous streams in a microfluidic channel. The laminar flow in the microchannel enables controlled diffusive mixing at the two liquid interfaces where the lipids self-assemble into vesicles. The liposomes formed by this self-assembly process are characterized using asymmetric flow field-flow fractionation combined with quasi-elastic light scattering and multiangle laser-light scattering. We observe that the vesicle size and size distribution are tunable over a mean diameter from 50 to 150 nm by adjusting the ratio of the alcohol-to-aqueous volumetric flow rate. We also observe that liposome formation depends more strongly on the focused alcohol stream width and its diffusive mixing with the aqueous stream than on the sheer forces at the solvent-buffer interface.

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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 À1 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.