Microfluidic Directed Formation of Liposomes of Controlled Size

Abstract

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.

Microfluidic hydrodynamic focusing based synthesis of POPC liposomes for model biological systems

“…Preparation of liposomes by microfluidic hydrodynamic focusing was conducted in a microchannel, following the procedure developed by Jahn et al [25][26][27]. The microchannel network was etched in a glass wafer and consisted of three inlet channels and one outlet, as illustrated in Figure 1.…”

“…The flow rate ratio (FRR) is often used as a means of varying vesicle size, including for DMPC vesicles [25][26][27]31]. We have, therefore, investigated here the effect of FRR for POPC liposomes.…”

“…More recently, a new approach based on hydrodynamic focusing in micro-channels [24] has been developed and successfully applied in synthesis of vesicles of various lipids, as well as other types on nanoparticles [25][26][27][28][29][30]. Microfluidic hydrodynamic focusing (MHF) is conducted by introducing the lipid solution through a central inlet channel of a microfluidic device, and focusing this central stream by the flow of an aqueous solution through two or more side channels [24].…”

“…Virtually simultaneous self-assembly into bilayers and deformation of planar bilayers into spherical vesicles ensures that the majority of synthesized liposomes are unilamellar. It was also shown that the size distribution of liposomes can be relatively easily controlled by changing the flow rates of the lipid solution in organic solvent and the aqueous phase [25,26], thus making it a relatively simple and straightforward method for production of unilamellar liposomes of controllable size distribution. Several studies have also used the MHF approach to encapsulate different molecules in the aqueous interior of liposomes [25][26][27][28][29], demonstrating its applicability in drug delivery and similar pharmaceutical and medical applications.…”

“…Among them, POPC is the most abundant [35][36][37] and has been extensively used as a model compound for representation of natural PC mixtures [38]. While the MHF method has been used in synthesis of liposomes with other PC lipids [25][26][27]31], these studies have used molecules with two identical saturated fatty acid chains attached to the glycerol backbone, whereas lipid molecules of natural membranes usually contain two different fatty acids [39]. Fatty acid chains of the POPC molecule are different in length with one of them also containing an unsaturated C=C bond which affects fluidity and other physical properties of the lipid bilayer.…”