All-Aqueous Assemblies via Interfacial Complexation: Toward Artificial Cell and Microniche Development.

In nature, the environment surrounding biomolecules and living cells can dictate their structure, function, and properties. Confinement is a key means to define and regulate such environments. For example, the confinement of appropriate constituents in compartments facilitates the assembly, dynamics, and function of biochemical machineries as well as subcellular organelles. Membraneless organelles, in particular, are thought to form via thermodynamic cues defined within the interior space of cells. On larger length scales, the confinement of living cells dictates cellular function for both mammalian and bacterial cells. One promising class of artificial structures that can recapitulate these multiscale confinement effects is based on aqueous two-phase systems (ATPSs). This feature article highlights recent developments in the production and stabilization of ATPS-droplet-based systems, with a focus on interfacial complexation. These systems enable structure formation, modulation, and triggered (dis)assembly, thereby allowing structures to be tailored to fit the desired function and designed for particular confinement studies. Open issues for both synthetic cells and niche studies are identified.

AWE-somes: All Water Emulsion Bodies with Permeable Shells and Selective Compartments.

Living cells exploit compartmentalization within organelles to spatially and temporally control reactions and pathways. Here, we use the all aqueous two phase system (ATPS) of poly(ethylene glycol) (PEG) and dextran to develop all water emulsion bodies, AWE-somes, a new class of encapsulated double emulsions as potential cell mimics. AWE-somes feature rigid polyelectrolyte (PE)/nanoparticle (NP) shells and double emulsion interiors. The shells form via complexation of PE and NP at interfaces of ATPS. The NPs, excluded from the drop phase, create an osmotic stress imbalance that removes water from the encapsulated phase and draws droplets of external PEG phase into the shells to form the double emulsion interior. We demonstrate that molecules can permeate the AWE-some shells, selectively partition into the internal droplets, and undergo reaction. AWE-somes have significant potential for creating functional, biocompatible protocell systems.

Tuning interfacial complexation in aqueous two phase systems with polyelectrolytes and nanoparticles for compound all water emulsion bodies (AWE-somes).

Interfacial complexation between two oppositely charged polymers in aqueous two phase systems (ATPSs) leads to the formation of mechanically robust microcapsules that can be stressed without losing their structural integrity. When a polyelectrolyte (PE) is replaced with a charged nanoparticle (NP), microcapsules with internal compartments can be generated within an encapsulated shell comprising NPs and PEs, named AWE-somes. These shells, made by interfacial complexation between PEs and NPs, are, however, very brittle and can lose their integrity under mechanical stress, potentially limiting their applications. Improved control over the properties and structure of microcapsules over a wide range is needed to enable their broad utilization. In this work, we show that interfacial complexation of a polycation with a mixture of a polyanion and a negatively charged NP in ATPS presents a simple yet versatile method of tuning the structure and properties of microcapsules. We show that internal structure, along with the mechanical robustness and stimuli-responsive properties of microcapsules, can be varied by changing the concentrations of polyanion and NP present in one of the two aqueous phases. Interfacial complexation of PE with mixtures of PE and NP provides a new strategy for controlling and imparting the properties and functionality of AWE-some interfacial membranes for applications in encapsulation and release of active agents and recapitulation of basic functions of living cells.

One-Step Generation of Cell-Encapsulating Compartments via Polyelectrolyte Complexation in an Aqueous Two Phase System.

Diverse fields including drug and gene delivery and live cell encapsulation require biologically compatible encapsulation systems. One widely adopted means of forming capsules exploits cargo-filled microdroplets in an external, immiscible liquid phase that are encapsulated by a membrane that forms by trapping of molecules or particles at the drop surface, facilitated by the interfacial tension. To eliminate the potentially deleterious oil phase often present in such processes, we exploit the aqueous two phase system of poly(ethylene glycol) (PEG) and dextran. We form capsules by placing dextran-rich microdroplets in an external PEG-rich phase. Strong polyelectrolytes present in either phase form complexes at the drop interface, thereby forming a membrane encapsulating the fluid interior. This process requires considerable finesse as both polyelectrolytes are soluble in either the drop or external phase, and the extremely low interfacial tension is too weak to provide a strong adsorption site for these molecules. The key to obtaining microcapsules is to tune the relative fluxes of the two polyelectrolytes so that they meet and complex at the interface. We identify conditions for which complexation can occur inside or outside of the drop phase, resulting in microparticles or poor encapsulation, respectively, or when properly balanced, at the interface, resulting in microcapsules. The resulting microcapsules respond to the stimuli of added salts or changes in osmotic pressure, allowing perturbation of capsule permeability or triggered release of capsule contents. We demonstrate that living cells can be sequestered and interrogated by encapsulating Pseudomonas aeruginosa PAO1 and using a Live/Dead assay to assess their viability. This method paves the way to the formation of a broad variety of versatile functional membranes around all aqueous capsules; by tuning the fluxes of complexing species to interact at the interface, membranes comprising other complexing functional moieties can be formed.

Trapping and assembly of living colloids at water-water interfaces.

We study the assembly of inert and living colloids in a two-phase water-water system that provides an environment that can sustain bacteria, providing a new structure with rich potential to confine and structure microbial communities. The water-water system, formed via phase separation of a casein and xanthan mixture, forms a 3-D structure of coexisting casein-rich and xanthan-rich phases. Fluorescent labelling and confocal microscopy reveal the attachment of these living colloids, including Escherichia coli and Pseudomonas aeruginosa, at the interface between the two phases. Inert colloids also become trapped at the interfaces, suggesting that the observed attachment can be attributed to capillarity. Over time, these structures coarsen and eventually degrade, illustrating the dynamic nature of these systems. This system lays the foundation for future studies of the interplay of physicochemical properties of the fluid interfaces and bulk phases and microbial responses they provoke to induce complex spatial organization, to study species which occupy distinct niches, and to optimize efficient microbial cross-feeding or protection from competitors.

Size evolution of highly amphiphilic macromolecular solution assemblies via a distinct bimodal pathway.

The solution self-assembly of macromolecular amphiphiles offers an efficient, bottom-up strategy for producing well-defined nanocarriers, with applications ranging from drug delivery to nanoreactors. Typically, the generation of uniform nanocarrier architectures is controlled by processing methods that rely on cosolvent mixtures. These preparation strategies hinge on the assumption that macromolecular solution nanostructures are kinetically stable following transfer from an organic/aqueous cosolvent into aqueous solution. Herein we demonstrate that unequivocal step-change shifts in micelle populations occur over several weeks following transfer into a highly selective solvent. The unexpected micelle growth evolves through a distinct bimodal distribution separated by multiple fusion events and critically depends on solution agitation. Notably, these results underscore fundamental similarities between assembly processes in amphiphilic polymer, small molecule and protein systems. Moreover, the non-equilibrium micelle size increase can have a major impact on the assumed stability of solution assemblies, for which performance is dictated by nanocarrier size and structure.