Monodisperse Uni- and Multicompartment Liposomes

Abstract

Liposomes are self-assembled phospholipid vesicles with great potential in fields ranging from targeted drug delivery to artificial cells. The formation of liposomes using microfluidic techniques has seen considerable progress, but the liposomes formation process itself has not been studied in great detail. As a result, high throughput, high-yielding routes to monodisperse liposomes with multiple compartments have not been demonstrated. Here, we report on a surfactant-assisted microfluidic route to uniform, single bilayer liposomes, ranging from 25 µm to 190 µm, and with or without multiple inner compartments. The key of our method is the precise control over the developing interfacial energies of complex W/O/W emulsion systems during liposome formation, which is achieved via an additional surfactant in the phase. The liposomes consist of single bilayers, as demonstrated by nanopore formation experiments and confocal fluoresce microscopy, and they can act as compartments for cell-free gene expression. The microfluidic technique can be expanded to create liposomes with a multitude of coupled compartments, opening routes to networks of multistep microreactors.There has been a significant interest in the use of liposomes, self-assembled phospholipid vesicles composed of bilayer membranes, in fields as diverse as targeted drug delivery, 1,2 membrane protein science, 3-5 bioreactors 6-8 and biosensors. 9,10 Cell-sized liposomes that encapsulate biomolecules and incorporate biological functions provide a versatile mimic of certain aspects of living cells, as exemplified by work showing RNA replication, [11][12][13] in vitro transcription and translation of gene networks, 6,14-16 and organization of cell division machinery in liposomes. [17][18][19][20][21][22][23][24]

Microfluidic Formation of Monodisperse Coacervate Organelles in Liposomes

“…Recently, microfluidic approaches to multicompartment10 and core–shell vesicle structures11 have been reported as advanced artificial cell models 10a, 11a. Herein, we demonstrate the embedding of coacervate droplets into liposomes.…”

“…Our method is flexible and highly controlled and produces monodisperse structures in high throughput (as high as 10 3  s −1 ) and high yields (more than 90 % of double emulsion droplets form liposomes) 10a. As Figure 1 d shows, both liposomes and inner coacervate droplets show high monodispersity; their mean diameters are, respectively, 78 μm and 10 μm and the coefficients of variation are 4 % and 11 %, respectively (Supporting Information, Figure S9).…”

“…In a typical experiment, we used a mixture of chloroform and hexane (30:70, v/v) containing 5.0 mg mL −1 l ‐α‐ phosphatidylcholine (egg PC) as the middle oil phase (O) and an aqueous solution of 10.0 wt% PVA and 0.3 wt% F‐68 as the outer water phase (W2, see the Supporting Information for full details) 10a. As the solvents evaporated, the as‐prepared double emulsion templates underwent a dewetting transition, generating uniform unilamellar liposomes (Figure 1 b2 and Supporting Information, Figure S6) 10a. Meanwhile, the encapsulated coacervate droplets coalesced into a single large droplet within approximately 15 min (Figure 1 c, Movie S2).…”

Droplet Microfluidics as a Tool for the Generation of Granular Matters and Functional Emulsions

“…In complex emulsions, such as double emulsions in which the droplets encapsulate yet more droplets in themselves, it is important to use proper sets of additives for each phase (surfactants, osmotic agents) in order to balance all types of forces in the system, including osmotic pressure, Laplace pressure and interfacial forces (Deng et al, 2016;Kanouni et al, 2002;Zinchenko et al, 2014).…”

“…The thickness of the shell can be further reduced by post-processing to submicron dimensions, i.e. squeezing the double emulsion through the narrow slit that causes the multiple emulsion to lose some shell volume (Akamatsu et al, 2015;Arriaga et al, 2015;Vian et al, 2016) or by dewetting of the shell phase which may lead to the formation of polymerosome (Deng et al, 2016;Ho et al, 2008;Kim et al, 2013).…”