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.

Figure 1

Hierarchical self-assembly of a terpolymer-stabilized coacervate protocell. Oppositely charged amylose biopolymers undergo complex coacervation and droplet formation, followed by interfacial self-assembly of terpolymer 1. (A) Confocal micrograph of terpolymer/coacervate protocells with internalized BSA-FITC (purple) and terpolymer membrane (green, Nile Red). (B) 3D representation of interfacial assembly of terpolymers.

Figure 2

Membrane stabilization and mixing study. Populations of coacervate droplets containing either BSA-FITC (green) or BSA-Cy5 (blue) were mixed after treatment with different amounts of terpolymer 1; the presence of a membrane was visualized with Nile Red (red). (A) Without terpolymer addition unstable coacervates mix immediately. Terpolymer addition of (B) 333 and (C) 1500 μg/mL resulted in slowed mixing or nonmixing, respectively (scale bars = 10 μm).

Figure 3

Demonstration of chemical communication between protocell populations. (A) Representation of the GOX/HRP enzyme cascade encapsulated in two protocell subpopulations, identified by FITC (green) or Cy5 (blue), respectively, with H₂O₂ being the content of this molecular conversation. (B) Example of confocal data obtained; resorufin (red) is produced preferentially in HRP-protocells changing them from blue to purple (scale bars = 20 μm). (C) Analysis of the average resorufin fluorescence in subpopulations of protocells: (i) Background levels of resorufin production were measured using GOX-protocells only, HRP-protocells only, or a mixture without glucose addition as controls (green half-spheres, blue half-spheres, or crosses, respectively). (ii) Increasing resorufin fluorescence in either GOX- or HRP-protocells in a mixed system (green or blue spheres, respectively). (iii) Increasing resorufin fluorescence in co-encapsulated GOX/HRP-protocells mixed with empty protocells (filled or empty triangles, respectively).

Figure 4

Three separate protocell populations encapsulating fluorescently labeled BSA with either FITC (green), Cy5 (blue), or RITC (red), persistent for 2.5 h after mixing (scale bar = 20 μm).