Hierarchical Self-Assembly of a Copolymer-Stabilized Coacervate Protocell

Abstract

Complex coacervate microdroplets are finding increased utility in synthetic cell applications due to their cytomimetic properties. However, their intrinsic membrane-free nature results in instability that limits their application in protocell research. Herein, we present the development of a new protocell model through the spontaneous interfacial self-assembly of copolymer molecules on biopolymer coacervate microdroplets. This hierarchical protocell model not only incorporates the favorable properties of coacervates (such as spontaneous assembly and macromolecular condensation) but also assimilates the essential features of a semipermeable copolymeric membrane (such as discretization and stabilization). This was accomplished by engineering an asymmetric, biodegradable triblock copolymer molecule comprising hydrophilic, hydrophobic, and polyanionic components capable of direct coacervate membranization via electrostatic surface anchoring and chain self-association. The resulting hierarchical protocell demonstrated striking integrity as a result of membrane formation, successfully stabilizing enzymatic cargo against coalescence and fusion in discrete protocellular populations. The semipermeable nature of the copolymeric membrane enabled the incorporation of a simple enzymatic cascade, demonstrating chemical communication between discrete populations of neighboring protocells. In this way, we pave the way for the development of new synthetic cell constructs.

Stabilization of All‐in‐Water Emulsions To Form Capsules as Artificial Cells

“…[23] The other way for ac hemical to adsorb at the interface is to designi ts uch that am oiety of this chemical can interactw ith the inner dropletc omponents, whereas another partc an interact with the outer continuousp hase. [22] Af atty acid bilayer has also been deposited at the surface of complex coacervate droplets throughe lectrostatic interactions between the inner dropletc omponents and the fattya cidc arboxylate groups. [47] Later,asimilar system was exploited to form bioreactors.…”

“…[14] These systems are attracting growingi nterest in the field of synthetic biology and in materials chemistry. [16][17][18][19][20][21][22][23] ATPS droplets have been developed to mimic organelles or protocells;h owever,t hey lack amembrane, whichisnecessary to control entry/exit of materials from compartments. [1,2,15] In theseA TPSs, the droplets that form upon stirring the solution coalescew ith time (hours) to yield am acroscopic liquid-liquidp hase separation, but recent investigations have shown numerousw ays to stabilize the dropletinterface, preventing coalescence.…”

Preparation of Swellable Hydrogel‐Containing Colloidosomes from Aqueous Two‐Phase Pickering Emulsion Droplets

“…[2,16,17] Moreover,c ross-linking beads using glutaraldehyde or derivatives [1] should also allow the production of colloidosomes in such ATPS.Anadditional advantage of the colloidosomes described herein compared to those prepared from conventional w/o emulsions,isthat they can spontaneously uptake certain payloads after their formation. [2,16,17] Moreover,c ross-linking beads using glutaraldehyde or derivatives [1] should also allow the production of colloidosomes in such ATPS.Anadditional advantage of the colloidosomes described herein compared to those prepared from conventional w/o emulsions,isthat they can spontaneously uptake certain payloads after their formation.…”

“…During the cross-linking process,t he cross-linkers may diffuse and react with the encapsulated biomolecules.M oreover, transfer of colloidosomes into water usually involves the use of organic solvents to remove the continuous oil phase. [2,9,16,17] However,t hese are not stable,b eing disassembled upon dilution below the concentration of ATPS formation. [9] In principle,w /w emulsions produced by liquid-liquid phase separation [10] can be used to uptake payloads because the resulting droplets can spontaneously sequester biomolecules.…”

“…[17,19] We used this technology to produce isolated hybrid hydrogelcontaining colloidosomes within w/o droplets in ah ighly controlled manner.T he w/w droplets (Dex/PEG) covered by LBs were produced in 40 pL w/o droplets and synthesis of PA A/dextran hydrogelled colloidosomes occurred in situ upon addition of both PA Aa nd EdC (Supporting Information, Figure S5). Droplet-based microfluidics has been widely used to increase the monodispersity of compartments prepared from hydrogels and ATPS.…”