Abstract:Coacervates have been widely studied as model compartments in protocell research. Complex coacervates composed of disordered proteins and RNAh ave also been shown to play an important role in cellular processes.H erein, we report on am icrofluidic strategy for constructing monodisperse coacervate droplets encapsulated within uniform unilamellar liposomes.T hese structures represent ab ottomup approach to hierarchically structured protocells,a sd emonstrated by storage and release of DNAfrom the encapsulated coacervates as well as localized transcription.Complex coacervation is the associative phase separation of oppositely charged polyelectrolytes.[1] This form of liquidliquid phase separation is apowerful means of compartmentalization and has been explored extensively as ap rotocell model for the construction of artificial cells or organelles. [2] Protocells based on simple and complex coacervates have been shown to display interesting properties,i ncluding enhanced enzyme catalysis, [3] selective partitioning of biomolecules, [4] and model crowded environments. [3e, 5] Importantly,c oacervates can also be found in living cells,f or example Pg ranules, [6] stress granules, [7] and Cajal bodies.[8]Increasing evidence suggests that these compartments originate via liquid-liquid phase separation (LLPS) of (intrinsically disordered) proteins and RNA [9] and play an important role in cell structure and functions involving RNAm etabolism.[9] Thep revalence and importance of coacervates in biology,i nc ombination with their relevance as artificial compartments,i nspired us to explore the formation of welldefined functional coacervates encapsulated within membranous structures.Recently,m icrofluidic approaches to multicompartment [10] and core-shell vesicle structures [11] have been reported as advanced artificial cell models.[10a, 11a] Herein, we demonstrate the embedding of coacervate droplets into liposomes.T hese internal organelle-like compartments allow for ahigh level of control over the spatial organization of biochemical processes.Similar liposomes containing asynthetic polymer-based aqueous two phase system (ATPS), that is,p olyethylene glycol and dextran (PEG-DEX), [12] have already been reported through abulk hydration of dried lipid membranes and shown diverse cell-like properties,s uch as microcompartmentalization, [12a,b] protein relocalization in response to stimuli, [12c] and asymmetric vesicle division.[12d]However,m ore bio-relevant coacervate systems have not been achieved in phospholipid vesicles until now,p robably because complex coacervates are highly charged, which interferes with conventional liposome preparation methods.[13] Additionally,t ypical liposome formation methods lead to polydisperse structures,g ive low yields,a nd show inefficient encapsulation. Furthermore,A TPS phase transitions often require undesired heating and cooling steps (the temperature applied is as high as 50 8 8C) [12a] or osmotic shocks, [14] which are incompatible with biological processes and cause ...
