Abstract

Cells, the discrete living systems that comprise all life on Earth, are a boundless source of inspiration and motivation for many researchers in the natural sciences. In the field of bottom-up synthetic cells, researchers seek to create multifaceted, self-assembled, chemical systems that mimic the properties and behaviours of natural life. In this perspective, we will describe the relatively recent application of complex coacervates to synthetic cells, and how they have been used to model an expanding range of biologically relevant phenomena. Furthermore, we will explore the unique advantages and disadvantages of coacervate-based synthetic cells, and their potential impact on the field in the years to come.

The construction of bottom-up synthetic cells has significantly matured over the last 20 years, and it is now possible to mimic some of the hallmarks of living systems reliably [1], such as metabolism, storage and manipulation of genetic information [2], and compartmentalization. The last of these, compartmentalization, has been thoroughly explored via a plethora of different synthetic capsule-based constructs, such as liposomes [3], polymersomes [4], proteinosomes [5], and colloidosomes [6,7]. While these systems have proven to be easily accessible and amenable to the incorporation of functional elements into discrete protocells, they often share the same drawback. Their inner environment is typically a dilute solution of functional macromolecules (enzymes, DNA) — a far cry from the crowded, highly concentrated, chemically complex environment found in the cell cytosol. Coacervation, or liquid–liquid phase separation, provides a synthetically simple and easy to deploy mimic of the cell cytosol (Table 1). Coacervates are formed either from the association of macromolecules via attractive forces (electrostatic, hydrophobic interactions) into metastable polymer-rich droplets, or from the non-association of incompatible aqueous polymers (such as PEG and dextran) into distinct polymer-rich phases [8]. While liquid–liquid phase separation in cells, as observed in membrane-less organelles, is also rapidly gaining significance in extant biology [911], coacervates have primarily been utilised by the synthetic cell community for their structural features and ability to effectively sequester functional materials [1214]. In this perspective, we describe some of the most recent examples of coacervate-based protocells, while also highlighting the main drawbacks that coacervates face. Finally, we will outline some of the pressing research questions that remain unanswered, and provide a vision for the future of coacervates in synthetic cell applications.

Table 1.
Overview of coacervate formation and properties
Formation Condensed phases form via the phase separation of incompatible aqueous polymers (such as PEG + Dextran) or the complexation of polyelectrolytes (such as ATP + Poly(lysine)) 
Physical Properties Size: 1–100 µm. Aqueous droplets with no interface/membrane that rapidly coalesce. Highly viscous and crowded microenvironments which still permit enzymatic processes and the diffusion of small molecules. 
Chemical Properties Coacervation (especially for complex coacervates) is strongly dependant on solution pH and ionic strength. Chemical properties depend on polymers utilised (thus highly tuneable). 
Characterisation Cell-sized, so typically via optical microscopy. 
Formation Condensed phases form via the phase separation of incompatible aqueous polymers (such as PEG + Dextran) or the complexation of polyelectrolytes (such as ATP + Poly(lysine)) 
Physical Properties Size: 1–100 µm. Aqueous droplets with no interface/membrane that rapidly coalesce. Highly viscous and crowded microenvironments which still permit enzymatic processes and the diffusion of small molecules. 
Chemical Properties Coacervation (especially for complex coacervates) is strongly dependant on solution pH and ionic strength. Chemical properties depend on polymers utilised (thus highly tuneable). 
Characterisation Cell-sized, so typically via optical microscopy. 

A surprisingly broad range of interesting biological phenomena can already be reconstituted within the confines of coacervate-based protocells (Figure 1). This is due to their tendency to accumulate all manner of functional macromolecules and their general biocompatibility, as they are often comprised of biologically derived materials (e.g. proteins, (oligo)nucleotides, polysaccharides). To start with, as the maintenance of genetic information is widely recognised as one of the prerequisites for living systems [15], it is comes as no surprise that the incorporation of information-processing machinery within coacervate-based protocells has already been well investigated [8]. Take, for example, the work of Keating and Bevilacqua on the catalytic activity of ribozymes, which displayed enhanced reaction rates within both associative [16,17] and non-associative [18,19] coacervates. Coacervates are also able to support in vitro transcription-translation (IVTT). Utilising polysaccharide/polypeptide coacervates, it was possible to produce a fluorescent protein mCherry, albeit with low yields, which was attributed to protein aggregation [20]. In another approach, a microfluidic setup was used to contain the IVTT reaction within coacervates formed in water-in-oil droplets, which demonstrated that the crowded environment not only supported IVTT, but resulted in increased transcription rates [21].

An overview of the current state of the art in coacervate-based protocells.

Figure 1.
An overview of the current state of the art in coacervate-based protocells.

The coacervate depicted here is an associative (complex) coacervate for clarity.

Figure 1.
An overview of the current state of the art in coacervate-based protocells.

The coacervate depicted here is an associative (complex) coacervate for clarity.

The ability of coacervate-based protocells to sequester materials not only applies to small molecules and enzymes as in the case of IVTT, but can also be extended to larger, self-contained synthetic systems, creating sub-compartmentalised protocells which have been designed to recreate the structural organization of organelles in eukaryotic cells. We have recently developed such a system, where an increase in enzymatic rates and protection from proteolytic degradation were demonstrated by the incorporation of polymersomes loaded with functional enzymatic cargo into the coacervate core [22]. Hybrid systems are also possible, such as the incorporation of intact, functional biological organelles (chloroplasts) into coacervate protocells [23]. Coacervates have also been used as sub-compartments in liposomes [24] and proteinosomes [25].

Surface tension dictates that the majority of coacervate-based protocells are spherical. As a plethora of cells adopt non-spherical morphologies to adapt to their environment or perform a specific function, engineering other, non-spherical protocell morphologies is thus an interesting challenge. So far, only a handful of studies have demonstrated the capability of coacervates to alter their shape. In an elegant example, Mann and co-workers developed a coacervate containing dipeptide progelators, which underwent a pH-induced transformation from spherical droplets to fibres, resulting in a hydrogel [26]. In a bioinspired approach, coacervates loaded with FtsZ, a protein involved in the bacterial division, were found to be capable of size and shape transformations [27]. It is also possible to utilise external forces to induce morphological transitions, such as the acoustic patterning [28] of coacervates undertaken in the Mann group [29].

One of the main limitations of using coacervates over membrane-bound protocell models is their inherent structural instability, as coacervate droplets are prone to spontaneous coalescence. Several strategies have been developed to combat this behaviour, for example, by coating coacervate droplets with assemblies of phospholipids [19,3032], or by alginate/gelatin-mediated in situ hydrogelation [33]. Our own contribution to this challenge utilises a polymer-mediated stabilisation methodology, where membranisation was achieved using a carefully designed block terpolymer that interacts electrostatically with amylose-based coacervate droplets [34]. The resultant protocells are remarkably stable under aqueous buffer conditions and mechanical manipulation, while still preserving a semi-permeable shell that allows trans-membrane diffusion of a wide range of molecular species [35].

All the above examples share a common theme — they all exploit the unique strength of coacervate-based synthetic cells to provide a structural scaffold within which a high concentration of functional cargo can be loaded. This advantage will inevitably be pressed in the coming years to develop increasingly complex life-like systems. However, it is important to note some of the inherent limitations of coacervates, and some of the challenges to be overcome in the field. First, while the structure of coacervates is much closer in charge density and viscosity to the cell cytosol than membrane-bound protocells, the chemical complexity seen in living cytosolic environments is impossible to emulate with a two-component mixture of macromolecules that do not contribute functionally and which cannot be reproduced for the generation of progeny. In this respect, coacervates will possibly outlive their usefulness in the pursuit of a truly bottom-up synthetic cell. It may become increasingly difficult to load coacervates with the enormous variety of molecules required for life, thus, a key challenge will be the engineering of coacervates capable of responding to a large influx of biomolecules. In one possible scenario, the coacervate phase could be utilised to accumulate material, template specific structural elements and act as an incubator for the synthetic cell, after which the coacervate dissipates or is consumed as a fuel by the nascent protocell. Similarly, coacervates could be important in providing an insight into the origin of life. Indeed, one of the earliest origin of life theories proposed by Oparin has coacervation at its core, where colloidal aggregates concentrated prebiotic components [36], and researchers continue to explore their role as an RNA concentrator in the RNA-world hypothesis [8].

On a related note, the vast majority of current coacervate protocells exist in a decidedly non-living, metastable or thermodynamically trapped state. The only exception, to our knowledge, is the engineering of an enzymatic cascade to provide spatiotemporal control over coacervate droplet formation by Spruijt and co-workers [37]. To be able to understand and mimic dynamic, far-from-equilibrium self-assembly in coacervate-based synthetic cells is an enormous challenge, as it represents life itself [38]. However, working in the favour of coacervates is their fundamental tuneability; afforded by virtually limitless chemical modification and combinations of different coacervate forming species.

In spite of these challenges, we believe that the current state of the art in coacervate protocells, where enzymatic processes are encapsulated in discrete, cytosol mimetic protocells, already has the potential to be developed into real-world applications. For example, coacervate protocells could be utilised in the drug discovery pipeline, where the effect of small molecule drugs on protein–protein interactions or membrane-bound proteins are assayed in a more cell-mimetic environment to provide more reliable scores before in vitro/in vivo studies. Continuing with a biomedical application, it would also be interesting to apply existing technology [39] to create 3D assemblages of coacervate protocells, towards the creation of synthetic tissues capable of loading high concentrations of functional cargo.

In our opinion, coacervates are an enticing protocell model, due principally to their unique ability to enhance their own functionality by the spontaneous uptake of functional materials. While challenges do exist for their continued development, they are not insurmountable and there will be enormous value in pursuing these systems towards the creation of more life-like protocells.

Abbreviations

     
  • IVTT

    in vitro transcription-translation

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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