Microfluidic Formation of Monodisperse Coacervate Organelles in Liposomes

Abstract

Coacervates have been widely studied as model compartments in protocell research. Complex coacervates composed of disordered proteins and RNA have also been shown to play an important role in cellular processes. Herein, we report on a microfluidic strategy for constructing monodisperse coacervate droplets encapsulated within uniform unilamellar liposomes. These structures represent a bottom-up approach to hierarchically structured protocells, as demonstrated by storage and release of DNA from the encapsulated coacervates as well as localized transcription.

Keywords: artificial organelles; coacervates; liposomes; microfluidics; phase transitions.

Figure 1

Encapsulation of coacervates into liposomes. a) Illustration and b) images of the microfluidic preparation of W/O/W double emulsions with coacervates as well as relevant dewetting transition and fusion process to form a liposome with a coacervate droplet. Inset in (b) shows the mixing of W1 and W1′ in microchannels. c,d) Confocal images of the fusion of small coacervates into a big coacervate in liposome (c) and as‐prepared uniform liposomes containing monodisperse coacervate droplets (d, panel d1 shows liposomes and residual oil droplets with excess lipids; panel d2 shows the labelled coacervates; panel d3 is the overlay of d1 and d2). Polycation=poly‐l‐lysine, polyanion=ATP.

Figure 2

Thermal dynamics of the membraneless organelle‐like compartment in liposomes. a) Illustration and b) confocal images of dissolution and coacervation of the coacervate droplets inside liposomes over time as temperature changes. c) Magnified views of a single liposome with dynamic artificial organelles and d) the relevant 3D fluorescence intensity profiles. Polycation=spermine, polyanion=polyU RNA.

Figure 3

Release and storage of labelled DNA molecules in artificial organelles. a) Illustration and b) confocal images show thermally triggered release and storage of labelled DNA molecules in the coacervates within liposomes. c) Kinetics of localized fluorescence of labelled DNA in liposomes and coacervate droplets when temperature was switched under or above coacervate LCST. Polycation=spermine, polyanion=polyU RNA.

Figure 4

Spatial organization of bio‐reaction in artificial organelles. a) Illustrations of IVTx in coacervate droplet in liposome and the working principle of detection of generated RNA using aptamer Spinach2 and dye DFHBI. b) Optical image of as‐formed liposomes containing coacervate droplet (Polycation=spermidine, polyanion=polyU RNA) and IVTx mix. c–e) Confocal images show RNA generation in coacervates over time.