Regulating Chemokine-Receptor Interactions through the Site-Specific Bioorthogonal Conjugation of Photoresponsive DNA Strands.

Oligonucleotide conjugation has emerged as a versatile molecular tool for regulating protein activity. A state-of-the-art labeling strategy includes the site-specific conjugation of DNA, by employing bioorthogonal groups genetically incorporated in proteins through unnatural amino acids (UAAs). The incorporation of UAAs in chemokines has to date, however, remained underexplored, probably due to their sometimes poor stability following recombinant expression. In this work, we designed a fluorescent stromal-derived factor-1ß (SDF-1ß) chemokine fusion protein with a bioorthogonal functionality amenable for click reactions. Using amber stop codon suppression, p-azido-L-phenylalanine was site-specifically incorporated in the fluorescent N-terminal fusion partner, superfolder green fluorescent protein (sfGFP). Conjugation to single-stranded DNAs (ssDNA), modified with a photocleavable spacer and a reactive bicyclononyne moiety, was performed to create a DNA-caged species that blocked the receptor binding ability. This inhibition was completely reversible by means of photocleavage of the ssDNA strands. The results described herein provide a versatile new direction for spatiotemporally regulating chemokine-receptor interactions, which is promising for tissue engineering purposes.

Confined Motion: Motility of Active Microparticles in Cell-Sized Lipid Vesicles.

Active materials can transduce external energy into kinetic energy at the nano and micron length scales. This unique feature has sparked much research, which ranges from achieving fundamental understanding of their motility to the assessment of potential applications. Traditionally, motility is studied as a function of internal features such as particle topology, while external parameters such as energy source are assessed mainly in bulk. However, in real-life applications, confinement plays a crucial role in determining the type of motion active particles can adapt. This feature has been however surprisingly underexplored experimentally. Here, we showcase a tunable experimental platform to gain an insight into the dynamics of active particles in environments with restricted 3D topology. Particularly, we examined the autonomous motion of coacervate micromotors confined in giant unilamellar vesicles (GUVs) spanning 10-50 µm in diameter and varied parameters including fuel and micromotor concentration. We observed anomalous diffusion upon confinement, leading to decreased motility, which was more pronounced in smaller compartments. The results indicate that the theoretically predicted hydrodynamic effect dominates the motion mechanism within this platform. Our study provides a versatile approach to understand the behavior of active matter under controlled, compartmentalized conditions.

DNA-Mediated Protein Shuttling between Coacervate-Based Artificial Cells.

The regulation of protein uptake and secretion is crucial for (inter)cellular signaling. Mimicking these molecular events is essential when engineering synthetic cellular systems. A first step towards achieving this goal is obtaining control over the uptake and release of proteins from synthetic cells in response to an external trigger. Herein, we have developed an artificial cell that sequesters and releases proteinaceous cargo upon addition of a coded chemical signal: single-stranded DNA oligos (ssDNA) were employed to independently control the localization of a set of three different ssDNA-modified proteins. The molecular coded signal allows for multiple iterations of triggered uptake and release, regulation of the amount and rate of protein release and the sequential release of the three different proteins. This signaling concept was furthermore used to directionally transfer a protein between two artificial cell populations, providing novel directions for engineering lifelike communication pathways inside higher order (proto)cellular structures.

Supramolecular Nanoscaffolds within Cytomimetic Protocells as Signal Localization Hubs.

The programmed construction of functional synthetic cells requires spatial control over arrays of biomolecules within the cytomimetic environment. The mimicry of the natural hierarchical assembly of biomolecules remains challenging due to the lack of an appropriate molecular toolbox. Herein, we report the implementation of DNA-decorated supramolecular assemblies as dynamic and responsive nanoscaffolds for the localization of arrays of DNA signal cargo within hierarchically assembled complex coacervate protocells. Protocells stabilized with a semipermeable membrane allow trafficking of single-stranded DNA between neighboring protocells. DNA duplex operations demonstrate the responsiveness of the nanoscaffolds to different input DNA strands via the reversible release of DNA cargo. Moreover, a second population of coacervate protocells with nanoscaffolds featuring a higher affinity for the DNA cargo enabled chemically programmed communication between both protocell populations. This combination of supramolecular structure and function paves the way for the next generation of protocells imbued with programmable, lifelike behaviors.

Physicochemical Characterization of Polymer-Stabilized Coacervate Protocells.

The bottom-up construction of cell mimics has produced a range of membrane-bound protocells that have been endowed with functionality and biochemical processes reminiscent of living systems. The contents of these compartments, however, experience semidilute conditions, whereas macromolecules in the cytosol exist in protein-rich, crowded environments that affect their physicochemical properties, such as diffusion and catalytic activity. Recently, complex coacervates have emerged as attractive protocellular models because their condensed interiors would be expected to mimic this crowding better. Here we explore some relevant physicochemical properties of a recently developed polymer-stabilized coacervate system, such as the diffusion of macromolecules in the condensed coacervate phase, relative to in dilute solutions, the buffering capacity of the core, the molecular organization of the polymer membrane, the permeability characteristics of this membrane towards a wide range of compounds, and the behavior of a simple enzymatic reaction. In addition, either the coacervate charge or the cargo charge is engineered to allow the selective loading of protein cargo into the coacervate protocells. Our in-depth characterization has revealed that these polymer-stabilized coacervate protocells have many desirable properties, thus making them attractive candidates for the investigation of biochemical processes in stable, controlled, tunable, and increasingly cell-like environments.

Development of Morphologically Discrete PEG-PDLLA Nanotubes for Precision Nanomedicine.

Precise control over the morphological features of nanoparticles is an important requisite for their application in nanomedical research. Parameters such as size and shape have been identified as critical features for effective nanotherapeutic technologies due to their role in circulation, distribution, and internalization in vivo. Tubular PEG-PDLLA polymersomes (nanotubes) exhibit an interesting morphology with potential for immunotherapeutics, as the elongated shape can affect cell-particle interactions. Developing methodologies that permit control over the precise form of such nanotubes is important for their biomedical implementation due to the stringent physicochemical constraints for efficacious performance. Through careful control over the engineering process, we demonstrate the generation of well-defined nanotubes based on polymersomes as small as 250 and 100 nm, which can be successfully shape transformed. The quality of the resulting nanostructures was established by physical characterization using AF4-MALS and cryo-TEM. Moreover, we show the successful loading of such nanotubes with model payloads (proteins and drugs). These findings provide a promising platform for implementation in biomedical applications in which discrete structure and functionality are essential features.

ATP-Mediated Transient Behavior of Stomatocyte Nanosystems.

In nature, dynamic processes are ubiquitous and often characterized by adaptive, transient behavior. Herein, we present the development of a transient bowl-shaped nanoreactor system, or stomatocyte, the properties of which are mediated by molecular interactions. In a stepwise fashion, we couple motility to a dynamic process, which is maintained by transient events; namely, binding and unbinding of adenosine triphosphate (ATP). The surface of the nanosystem is decorated with polylysine (PLL), and regulation is achieved by addition of ATP. The dynamic interaction between PLL and ATP leads to an increase in the hydrophobicity of the PLL-ATP complex and subsequently to a collapse of the polymer; this causes a narrowing of the opening of the stomatocytes. The presence of the apyrase, which hydrolyzes ATP, leads to a decrease of the ATP concentration, decomplexation of PLL, and reopening of the stomatocyte. The competition between ATP input and consumption gives rise to a transient state that is controlled by the out-of-equilibrium process.

Hierarchical Self-Assembly of a Copolymer-Stabilized Coacervate Protocell.

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.

Optimising the synthesis, polymer membrane encapsulation and photoreduction performance of Ru(II)- and Ir(III)-bis(terpyridine) cytochrome c bioconjugates.

Ruthenium(II) and iridium(III) bis(terpyridine) complexes were prepared with maleimide functionalities in order to site-specifically modify yeast iso-1 cytochrome c possessing a single cysteine residue available for modification (CYS102). Single X-ray crystal structures were solved for aniline and maleimide Ru(II) 3 and Ru(II) 4, respectively, providing detailed structural detail of the complexes. Light-activated bioconjugates prepared from Ru(II) 4 in the presence of tris(2-carboxyethyl)-phosphine (TCEP) significantly improved yields from 6% to 27%. Photoinduced electron transfer studies of Ru(II)-cyt c in bulk solution and polymer membrane encapsulated specimens were performed using EDTA as a sacrificial electron donor. It was found that membrane encapsulation of Ru(II)-cyt c in PS140-b-PAA48 resulted in a quantum efficiency of 1.1 ± 0.3 × 10(-3), which was a two-fold increase relative to the bulk. Moreover, Ir(III)-cyt c bioconjugates showed a quantum efficiency of 3.8 ± 1.9 × 10(-1), equivalent to a ∼640-fold increase relative to bulk Ru(II)-cyt c.

Toward Artificial Cell-Mediated Tissue Engineering: A New Perspective.

The fast-growing pace of regenerative medicine research has allowed the development of a range of novel approaches to tissue engineering applications. Until recently, the main points of interest in the majority of studies have been to combine different materials to control cellular behavior and use different techniques to optimize tissue formation, from 3-D bioprinting to in situ regeneration. However, with the increase of the understanding of the fundamentals of cellular organization, tissue development, and regeneration, has also come the realization that for the next step in tissue engineering, a higher level of spatiotemporal control on cell-matrix interactions is required. It is proposed that the combination of artificial cell research with tissue engineering could provide a route toward control over complex tissue development. By equipping artificial cells with the underlying mechanisms of cellular functions, such as communication mechanisms, migration behavior, or the coherent behavior of cells depending on the surrounding matrix properties, they can be applied in instructing native cells into desired differentiation behavior at a resolution not to be attained with traditional matrix materials.

Enzymatic Regulation of Protein-Protein Interactions in Artificial Cells.

Membraneless organelles are important for spatial organization of proteins and regulation of intracellular processes. Proteins can be recruited to these condensates by specific protein-protein or protein-nucleic acid interactions, which are often regulated by post-translational modifications. However, the mechanisms behind these dynamic, affinity-based protein recruitment events are not well understood. Here, a coacervate system that incorporates the 14-3-3 scaffold protein to study enzymatically regulated recruitment of 14-3-3-binding proteins is presented, which mostly bind in a phosphorylation-dependent manner. Synthetic coacervates are efficiently loaded with 14-3-3, and phosphorylated binding partners, such as the c-Raf pS233/pS259 peptide (c-Raf), show 14-3-3-dependent sequestration with up to 161-fold increase in local concentration. The c-Raf domain is fused to green fluorescent protein (GFP-c-Raf) to demonstrate recruitment of proteins. In situ phosphorylation of GFP-c-Raf by a kinase leads to enzymatically regulated uptake. The introduction of a phosphatase into coacervates preloaded with the phosphorylated 14-3-3-GFP-c-Raf complex results in a significant cargo efflux mediated by dephosphorylation. Finally, the general applicability of this platform to study protein-protein interactions is demonstrated by the phosphorylation-dependent and 14-3-3-mediated active reconstitution of a split-luciferase inside artificial cells. This work presents an approach to study dynamically regulated protein recruitment in condensates, using native interaction domains.

Terpolymer-stabilized complex coacervates: A robust and versatile synthetic cell platform.

The utilization of liquid-liquid phase separated systems has seen increased attention as synthetic cell platforms due to their innate ability to sequester interesting, functional, and biologically relevant materials. However, their applications are limited by the temporal stability of such condensed phases. While there are a number of strategies toward droplet stabilization, in our group we have developed a polymer-based approach to stabilize complex coacervate microdroplets. These protocells are remarkably robust and have been utilized to support a number of new protocellular applications. Here, we describe in detail the methodologies we have developed for the synthesis of the starting components, their formation into stable, cargo-loaded protocells, and how these protocells are treated post-formation to purify and analyze the resultant functional self-assembled systems.

Engineering of Biocompatible Coacervate-Based Synthetic Cells.

Polymer-stabilized complex coacervate microdroplets have emerged as a robust platform for synthetic cell research. Their unique core-shell properties enable the sequestration of high concentrations of biologically relevant macromolecules and their subsequent release through the semipermeable membrane. These unique properties render the synthetic cell platform highly suitable for a range of biomedical applications, as long as its biocompatibility upon interaction with biological cells is ensured. The purpose of this study is to investigate how the structure and formulation of these coacervate-based synthetic cells impact the viability of several different cell lines. Through careful examination of the individual synthetic cell components, it became evident that the presence of free polycation and membrane-forming polymer had to be prevented to ensure cell viability. After closely examining the structure-toxicity relationship, a set of conditions could be found whereby no detrimental effects were observed, when the artificial cells were cocultured with RAW264.7 cells. This opens up a range of possibilities to use this modular system for biomedical applications and creates design rules for the next generation of coacervate-based, biomedically relevant particles.

Engineering transient dynamics of artificial cells by stochastic distribution of enzymes.

Random fluctuations are inherent to all complex molecular systems. Although nature has evolved mechanisms to control stochastic events to achieve the desired biological output, reproducing this in synthetic systems represents a significant challenge. Here we present an artificial platform that enables us to exploit stochasticity to direct motile behavior. We found that enzymes, when confined to the fluidic polymer membrane of a core-shell coacervate, were distributed stochastically in time and space. This resulted in a transient, asymmetric configuration of propulsive units, which imparted motility to such coacervates in presence of substrate. This mechanism was confirmed by stochastic modelling and simulations in silico. Furthermore, we showed that a deeper understanding of the mechanism of stochasticity could be utilized to modulate the motion output. Conceptually, this work represents a leap in design philosophy in the construction of synthetic systems with life-like behaviors.

Programmed spatial organization of biomacromolecules into discrete, coacervate-based protocells.

The cell cytosol is crowded with high concentrations of many different biomacromolecules, which is difficult to mimic in bottom-up synthetic cell research and limits the functionality of existing protocellular platforms. There is thus a clear need for a general, biocompatible, and accessible tool to more accurately emulate this environment. Herein, we describe the development of a discrete, membrane-bound coacervate-based protocellular platform that utilizes the well-known binding motif between Ni2+-nitrilotriacetic acid and His-tagged proteins to exercise a high level of control over the loading of biologically relevant macromolecules. This platform can accrete proteins in a controlled, efficient, and benign manner, culminating in the enhancement of an encapsulated two-enzyme cascade and protease-mediated cargo secretion, highlighting the potency of this methodology. This versatile approach for programmed spatial organization of biologically relevant proteins expands the protocellular toolbox, and paves the way for the development of the next generation of complex yet well-regulated synthetic cells.

Tuning Size and Morphology of mPEG-b-p(HPMA-Bz) Copolymer Self-Assemblies Using Microfluidics.

The careful design of nanoparticles, in terms of size and morphology, is of great importance to developing effective drug delivery systems. The ability to precisely tailor nanoparticles in size and morphology during polymer self-assembly was therefore investigated. Four poly(ethylene glycol)-b-poly(N-2-benzoyloxypropyl methacrylamide) mPEG-b-p(HPMA-Bz) block copolymers with a fixed hydrophilic block of mPEG 5 kDa and a varying molecular weight of the hydrophobic p(HPMA-Bz) block (A: 17.1, B: 10.0, C: 5.2 and D: 2.7 kDa) were self-assembled into nanoparticles by nanoprecipitation under well-defined flow conditions, using microfluidics, at different concentrations. The nanoparticles from polymer A, increased in size from 55 to 90 nm using lower polymer concentrations and slower flow rates and even polymer vesicles were formed along with micelles. Similarly, nanoparticles from polymer D increased in size from 35 to 70 nm at slower flow rates and also formed vesicles along with micelles, regardless of the used concentration. Differently, polymers B and C mainly self-assembled into micelles at the different applied flow rates with negligible size difference. In conclusion, this study demonstrates that the self-assembly of mPEG-b-p(HPMA-Bz) block copolymers can be easily tailored in size and morphology using microfluidics and is therefore an attractive option for further scaled-up production activities.

Pathway dependent shape-transformation of azide-decorated polymersomes.

Here we report the shape transformation of poly(ethylene glycol)-polystyrene (PEG-PS) polymersomes into ordered inverse morphologies, directed by the salt concentration of the medium and the presence of azide groups on the polymersome surface. The azide moieties introduced at the chain ends of the PEG blocks induce a difference in hydrodynamic volume of the hydrophilic domains at the inner and outer side of the vesicular membrane, allowing control over its spontaneous curvature and hence the pathway of shape deformation. This simple modification enables access to intricate morphologies which are traditionally only accessible via the application of complex polymer building blocks.

Multifaceted cell mimicry in coacervate-based synthetic cells.

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.

Mimicking Cellular Compartmentalization in a Hierarchical Protocell through Spontaneous Spatial Organization.

A systemic feature of eukaryotic cells is the spatial organization of functional components through compartmentalization. Developing protocells with compartmentalized synthetic organelles is, therefore, a critical milestone toward emulating one of the core characteristics of cellular life. Here we demonstrate the bottom-up, multistep, noncovalent, assembly of rudimentary subcompartmentalized protocells through the spontaneous encapsulation of semipermeable, polymersome proto-organelles inside cell-sized coacervates. The coacervate microdroplets are membranized using tailor-made terpolymers, to complete the hierarchical self-assembly of protocells, a system that mimics both the condensed cytosol and the structure of a cell membrane. In this way, the spatial organization of enzymes can be finely tuned, leading to an enhancement of functionality. Moreover, incompatible components can be sequestered in the same microenvironments without detrimental effect. The robust stability of the subcompartmentalized coacervate protocells in biocompatible milieu, such as in PBS or cell culture media, makes it a versatile platform to be extended toward studies in vitro, and perhaps, in vivo.

The hallmarks of living systems: towards creating artificial cells.

Despite the astonishing diversity and complexity of living systems, they all share five common hallmarks: compartmentalization, growth and division, information processing, energy transduction and adaptability. In this review, we give not only examples of how cells satisfy these requirements for life and the ways in which it is possible to emulate these characteristics in engineered platforms, but also the gaps that remain to be bridged. The bottom-up synthesis of life-like systems continues to be driven forward by the advent of new technologies, by the discovery of biological phenomena through their transplantation to experimentally simpler constructs and by providing insights into one of the oldest questions posed by mankind, the origin of life on Earth.