A microfluidic platform for the synthesis of polymer and polymer-protein-based protocells.

In this study, we demonstrate the fabrication of polymersomes, protein-blended polymersomes, and polymeric microcapsules using droplet microfluidics. Polymersomes with uniform, single bilayers and controlled diameters are assembled from water-in-oil-in-water double-emulsion droplets. This technique relies on adjusting the interfacial energies of the droplet to completely separate the polymer-stabilized inner core from the oil shell. Protein-blended polymersomes are prepared by dissolving protein in the inner and outer phases of polymer-stabilized droplets. Cell-sized polymeric microcapsules are assembled by size reduction in the inner core through osmosis followed by evaporation of the middle phase. All methods are developed and validated using the same glass-capillary microfluidic apparatus. This integrative approach not only demonstrates the versatility of our setup, but also holds significant promise for standardizing and customizing the production of polymer-based artificial cells.

Synthetic control of actin polymerization and symmetry breaking in active protocells.

Nonlinear biomolecular interactions on membranes drive membrane remodeling crucial for biological processes including chemotaxis, cytokinesis, and endocytosis. The complexity of biomolecular interactions, their redundancy, and the importance of spatiotemporal context in membrane organization impede understanding of the physical principles governing membrane mechanics. Developing a minimal in vitro system that mimics molecular signaling and membrane remodeling while maintaining physiological fidelity poses a major challenge. Inspired by chemotaxis, we reconstructed chemically regulated actin polymerization inside vesicles, guiding membrane self-organization. An external, undirected chemical input induced directed actin polymerization and membrane deformation uncorrelated with upstream biochemical cues, suggesting symmetry breaking. A biophysical model incorporating actin dynamics and membrane mechanics proposes that uneven actin distributions cause nonlinear membrane deformations, consistent with experimental findings. This protocellular system illuminates the interplay between actin dynamics and membrane shape during symmetry breaking, offering insights into chemotaxis and other cell biological processes.

Synthetic Condensates and Cell-Like Architectures from Amphiphilic DNA Nanostructures.

Synthetic droplets and condensates are becoming increasingly common constituents of advanced biomimetic systems and synthetic cells, where they can be used to establish compartmentalization and sustain life-like responses. Synthetic DNA nanostructures have demonstrated significant potential as condensate-forming building blocks owing to their programmable shape, chemical functionalization, and self-assembly behavior. We have recently demonstrated that amphiphilic DNA "nanostars", obtained by labeling DNA junctions with hydrophobic moieties, constitute a particularly robust and versatile solution. The resulting amphiphilic DNA condensates can be programmed to display complex, multi-compartment internal architectures, structurally respond to various external stimuli, synthesize macromolecules, capture and release payloads, undergo morphological transformations, and interact with live cells. Here, we demonstrate protocols for preparing amphiphilic DNA condensates starting from constituent DNA oligonucleotides. We will address (i) single-component systems forming uniform condensates, (ii) two-component systems forming core-shell condensates, and (iii) systems in which the condensates are modified to support in vitro transcription of RNA nanostructures.

Cell-free Protein Synthesis System for Building Synthetic Cells.

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for the core machinery of the Central Dogma, standing as an optimal chassis for the integration and assembly of diverse artificial cellular mimicry systems. Despite its frequent use in the fabrication of synthetic cells, establishing a tailored and robust CFPS system for a specific application remains a nontrivial challenge. In this methods paper, we present a comprehensive protocol for the CFPS system, routinely employed in constructing synthetic cells. This protocol encompasses key stages in the preparation of the CFPS system, including the cell extract, template preparation, and routine expression optimization utilizing a fluorescent reporter protein. Additionally, we show representative results by encapsulating the CFPS system within various micro-compartments, such as monolayer droplets, double-emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we elucidate the critical steps and conditions necessary for the successful assembly of these CFPS systems in distinct environments. We expect that our approach will facilitate the establishment of good working practices among various laboratories within the continuously expanding synthetic cell community, thereby accelerating progress in the field of synthetic cell development.

Evaluation of polymersome permeability as a fundamental aspect towards the development of artificial cells and nanofactories.

Polymersomes are synthetic vesicles with potential use in healthcare, chemical transformations in confined environment (nanofactories), and in the construction of artificial cells and organelles. In this framework, one of the most important features of such supramolecular structures is the permeability behavior allowing for selective control of mass exchange between the inner and outer compartments. The use of biological and synthetic nanopores in this regard is the most common strategy to impart permeability nevertheless, this typically requires fairly complex strategies to enable porosity. Yet, investigations concerning the permeability of polymer vesicles to different analytes still requires further exploration and, taking these considerations into account, we have detailed investigated the permeability behavior of a variety of polymersomes with regard to different analytes (water, protons, and rhodamine B) which were selected as models for solvents, ions, and small molecules. Polymersomes based on hydrophilic blocks of poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) or PEO (poly(ethylene oxide)) linked to the non-responsive blocks poly[N-(4-isopropylphenylacetamide)ethyl methacrylate] (PPPhA) or poly(methyl methacrylate) (PMMA), or to the stimuli pH-responsive block poly[2-(diisopropylamino)ethyl methacrylate] (PDPA) have been investigated. Interestingly, the produced PEO-based vesicles are notably larger than the ones produced using PHPMA-containing block copolymers. The experimental results reveal that all the vesicles are inherently permeable to some extent with permeability behavior following exponential profiles. Nevertheless, polymersomes based on PMMA as the hydrophobic component were demonstrated to be the least permeable to the small molecule rhodamine B as well as to water. The synthetic vesicles based on the pH-responsive PDPA block exhibited restrictive and notably slow proton permeability as attributed to partial chain protonation upon acidification of the medium. The dye permeability was evidenced to be much slower than ion or solvent diffusion, and in the case of pH-responsive assemblies, it was demonstrated to also depend on the ionic strength of the environment. These findings are understood to be highly relevant towards polymer selection for the production of synthetic vesicles with selective and time-dependent permeability, and it may thus contribute in advancing biomimicry and nanomedicine.

Interface Binding Mechanism of Nanoclay Hybridized Coacervate Microdroplets for the Controllable Construction of Protocells.

Coacervate microdroplets, a protocell model in exploring the origin of life, have gained significant attention. Clay minerals, catalysts during the origin of life, are crucial in the chemical evolution of small molecules into biopolymers. However, our understanding of the relationship between clay minerals and the formation and evolution of protocells on early Earth remains limited. In this work, the nanoclay montmorillonite nanosheet (MMT-Na) was employed to investigate its interaction with coacervate microdroplets formed by oligolysine (K10) and adenine nucleoside triphosphate (ATP). As an anionic component, MMT-Na was noted to promote the formation of coacervate microdroplets. Furthermore, the efficiency of ssDNA enrichment and the degree of ssDNA hybridization within these microdroplets were significantly improved. By combining inorganic nanoclay with organic biopolymers, our work provides an efficient way to enrich genetic biomolecules in the primitive Earth environment and builds a nanoclay-based coacervate microdroplets, shedding new light on life's origin and protocell evolution.

Magnetic Modulation of Biochemical Synthesis in Synthetic Cells.

Synthetic cells can be constructed from diverse molecular components, without the design constraints associated with modifying 'living' biological systems. This can be exploited to generate cells with abiotic components, creating functionalities absent in biology. One example is magnetic responsiveness, the activation and modulation of encapsulated biochemical processes using a magnetic field, which is absent from existing synthetic cell designs. This is a critical oversight, as magnetic fields are uniquely bio-orthogonal, noninvasive, and highly penetrative. Here, we address this by producing artificial magneto-responsive organelles by coupling thermoresponsive membranes with hyperthermic Fe3O4 nanoparticles and embedding them in synthetic cells. Combining these systems enables synthetic cell microreactors to be built using a nested vesicle architecture, which can respond to alternating magnetic fields through in situ enzymatic catalysis. We also demonstrate the modulation of biochemical reactions by using different magnetic field strengths and the potential to tune the system using different lipid compositions. This platform could unlock a wide range of applications for synthetic cells as programmable micromachines in biomedicine and biotechnology.

Living cells and biological mechanisms as prototypes for developing chemical artificial intelligence.

Artificial Intelligence (AI) is having a revolutionary impact on our societies. It is helping humans in facing the global challenges of this century. Traditionally, AI is developed in software or through neuromorphic engineering in hardware. More recently, a brand-new strategy has been proposed. It is the so-called Chemical AI (CAI), which exploits molecular, supramolecular, and systems chemistry in wetware to mimic human intelligence. In this work, two promising approaches for boosting CAI are described. One regards designing and implementing neural surrogates that can communicate through optical or chemical signals and give rise to networks for computational purposes and to develop micro/nanorobotics. The other approach concerns "bottom-up synthetic cells" that can be exploited for applications in various scenarios, including future nano-medicine. Both topics are presented at a basic level, mainly to inform the broader audience of non-specialists, and so favour the rise of interest in these frontier subjects.

Switchable and orthogonal gene expression control inside artificial cells by synthetic riboswitches.

Here we report two novel synthetic riboswitches that respond to ASP2905 and theophylline and function in reconstituted cell-free protein synthesis (CFPS) system. We encapsulated the CFPS system as well as DNA-templated encoding reporter genes regulated by these orthogonal riboswitches inside liposomes, and achieved switchable and orthogonal control over gene expression by external stimulation with the cognate ligands.

Stabilization of Prebiotic Vesicles by Peptides Depends on Sequence and Chirality: A Mechanism for Selection of Protocell-Associated Peptides.

Cells require oligonucleotides and polypeptides with specific, homochiral sequences to perform essential functions, but it is unclear how such oligomers were selected from random sequences at the origin of life. Cells were probably preceded by simple compartments such as fatty acid vesicles, and oligomers that increased the stability, growth, or division of vesicles could have thereby increased in frequency. We therefore tested whether prebiotic peptides alter the stability or growth of vesicles composed of a prebiotic fatty acid. We find that three of 15 dipeptides tested reduce salt-induced flocculation of vesicles. All three contain leucine, and increasing their length increases the efficacy. Also, leucine-leucine but not alanine-alanine increases the size of vesicles grown by multiple additions of micelles. In a molecular simulation, leucine-leucine docks to the membrane, with the side chains inserted into the hydrophobic core of the bilayer, while alanine-alanine fails to dock. Finally, the heterochiral forms of leucine-leucine, at a high concentration, rapidly shrink the vesicles and make them leakier and less stable to high pH than the homochiral forms do. Thus, prebiotic peptide-membrane interactions influence the flocculation, growth, size, leakiness, and pH stability of prebiotic vesicles, with differential effects due to sequence, length, and chirality. These differences could lead to a population of vesicles enriched for peptides with beneficial sequence and chirality, beginning selection for the functional oligomers that underpin life.

Designing Configurable Soft Microgelsomes as a Smart Biomimetic Protocell.

Self-assembly is an intriguing aspect of primitive cells. The construction of a semipermeable compartment with a robust framework of soft material capable of housing an array of functional components for chemical changes is essential for the fabrication of synthetic protocells. Microgels, loosely cross-linked polymer networks, are suitable building blocks for protocell capsule generation due to their porous structure, tunable properties, and assembly at the emulsion interface. Here, we present an interfacial assembly of microgel-based microcompartments (microgelsomes, MGC) that are defined by a semipermeable, temperature-responsive elastic membrane formed by densely packed microgels in a monolayer. The water-dispersible microgelsomes can thermally shuttle between 10 and 95 °C while retaining their structural integrity. Importantly, the microgelsomes exhibited distinct properties of protocells, such as cargo encapsulation, semipermeable membrane, DNA amplification, and membrane-gated compartmentalized enzymatic cascade reaction. This versatile approach for the construction of biomimetic microcompartments augments the protocell library and paves the way for programmable synthetic cells.

Dipeptide coacervates as artificial membraneless organelles for bioorthogonal catalysis.

Artificial organelles can manipulate cellular functions and introduce non-biological processes into cells. Coacervate droplets have emerged as a close analog of membraneless cellular organelles. Their biomimetic properties, such as molecular crowding and selective partitioning, make them promising components for designing cell-like materials. However, their use as artificial organelles has been limited by their complex molecular structure, limited control over internal microenvironment properties, and inherent colloidal instability. Here we report the design of dipeptide coacervates that exhibit enhanced stability, biocompatibility, and a hydrophobic microenvironment. The hydrophobic character facilitates the encapsulation of hydrophobic species, including transition metal-based catalysts, enhancing their efficiency in aqueous environments. Dipeptide coacervates carrying a metal-based catalyst are incorporated as active artificial organelles in cells and trigger an internal non-biological chemical reaction. The development of coacervates with a hydrophobic microenvironment opens an alternative avenue in the field of biomimetic materials with applications in catalysis and synthetic biology.

Superstructural ordering in self-sorting coacervate-based protocell networks.

Bottom-up assembly of higher-order cytomimetic systems capable of coordinated physical behaviours, collective chemical signalling and spatially integrated processing is a key challenge in the study of artificial multicellularity. Here we develop an interactive binary population of coacervate microdroplets that spontaneously self-sort into chain-like protocell networks with an alternating sequence of structurally and compositionally dissimilar microdomains with hemispherical contact points. The protocell superstructures exhibit macromolecular self-sorting, spatially localized enzyme/ribozyme biocatalysis and interdroplet molecular translocation. They are capable of topographical reconfiguration using chemical or light-mediated stimuli and can be used as a micro-extraction system for macroscale biomolecular sorting. Our methodology opens a pathway towards the self-assembly of multicomponent protocell networks based on selective processes of coacervate droplet-droplet adhesion and fusion, and provides a step towards the spontaneous orchestration of protocell models into artificial tissues and colonies with ordered architectures and collective functions.

Cell Free Expression in Proteinosomes Prepared from Native Protein-PNIPAAm Conjugates.

Towards the goal of building synthetic cells from the bottom-up, the establishment of micrometer-sized compartments that contain and support cell free transcription and translation that couple cellular structure to function is of critical importance. Proteinosomes, formed from crosslinked cationized protein-polymer conjugates offer a promising solution to membrane-bound compartmentalization with an open, semi-permeable membrane. Critically, to date, there has been no demonstration of cell free transcription and translation within water-in-water proteinosomes. Herein, a novel approach to generate proteinosomes that can support cell free transcription and translation is presented. This approach generates proteinosomes directly from native protein-polymer (BSA-PNIPAAm) conjugates. These native proteinosomes offer an excellent alternative as a synthetic cell chassis to other membrane bound compartments. Significantly, the native proteinosomes are stable under high salt conditions that enables the ability to support cell free transcription and translation and offer enhanced protein expression compared to proteinosomes prepared from traditional methodologies. Furthermore, the integration of native proteinosomes into higher order synthetic cellular architectures with membrane free compartments such as liposomes is demonstrated. The integration of bioinspired architectural elements with the central dogma is an essential building block for realizing minimal synthetic cells and is key for exploiting artificial cells in real-world applications.

Biofunctional coacervate-based artificial protocells with membrane-like and cytoplasm-like structures for the treatment of persistent hyperuricemia.

Coacervate droplets formed by liquid-liquid phase separation have attracted considerable attention due to their ability to enrich biomacromolecules while preserving their bioactivities. However, there are challenges to develop coacervate droplets as delivery vesicles for therapeutics resulting from the lack of physiological stability and inherent lack of membranes in coacervate droplets. Herein, polylysine-polynucleotide complex coacervate droplets with favorable physiological stability are formulated to efficiently and facilely concentrate small molecules, biomacromolecules and nanoparticles without organic solvents. To improve the biocompatibility, the PEGylated phospholipid membrane is further coated on the surface of the coacervate droplets to prepare coacervate-based artificial protocells (ArtPC) with membrane-like and cytoplasm-like structures. The ArtPC can confine the cyclic catalytic system of uricase and catalase inside to degrade uric acid and deplete the toxicity of H2O2. This biofunctional ArtPC effectively reduces blood uric acid levels and prevents renal injuries in mice with persistent hyperuricemia. The ArtPC-based therapy can bridge the disciplines of synthetic biology, pharmaceutics and therapeutics.

Identifying Conditions for Protein Synthesis Inside Giant Vesicles Using Microfluidics toward Standardized Artificial Cell Production.

To expand the range of practical applications of artificial cells, it is important to standardize the production process of giant (cell-sized) vesicles that encapsulate reconstituted biochemical reaction systems. For this purpose, a rapidly developing microfluidics-based giant vesicle generation system is a promising approach, similar to the droplet assay systems that are already widespread in the market. In this study, we examined the composition of the solutions used to generate vesicles encapsulating the in vitro transcription-translation (IVTT) system. We show that tuning of the lipid composition and adding poly(vinyl alcohol) to the outer solution improved the stability of the transition process into the lipid membrane so that protein synthesis proceeded in vesicles. The direct integration of α-hemolysin nanopores synthesized in situ was also demonstrated. These protein-synthesizing monodisperse giant vesicles can be prepared by using a simple microfluidic fabrication/operation with a commercial IVTT system.

Organization of an Artificial Multicellular System with a Tunable DNA Patch on a Membrane Surface.

Coordinating multiple artificial cellular compartments into a well-organized artificial multicellular system (AMS) is of great interest in bottom-up synthetic biology. However, developing a facile strategy for fabricating an AMS with a controlled arrangement remains a challenge. Herein, utilizing in situ DNA hybridization chain reaction on the membrane surface, we developed a DNA patch-based strategy to direct the interconnection of vesicles. By tuning the DNA patch that generates heterotrophic adhesion for the attachment of vesicles, we could produce an AMS with higher-order structures straightforwardly and effectively. Furthermore, a hybrid AMS comprising live cells and vesicles was fabricated, and we found the hybrid AMS with higher-order structures arouses efficient molecular transportation from vesicles to living cells. In brief, our work provides a versatile strategy for modulating the self-assembly of AMSs, which could expand our capability to engineer synthetic biological systems and benefit synthetic cell research in programmable manipulation of intercellular communications.

Selective amide bond formation in redox-active coacervate protocells.

Coacervate droplets are promising protocell models because they sequester a wide range of guest molecules and may catalyze their conversion. However, it remains unclear how life's building blocks, including peptides, could be synthesized from primitive precursor molecules inside such protocells. Here, we develop a redox-active protocell model formed by phase separation of prebiotically relevant ferricyanide (Fe(CN)63-) molecules and cationic peptides. Their assembly into coacervates can be regulated by redox chemistry and the coacervates act as oxidizing hubs for sequestered metabolites, like NAD(P)H and gluthathione. Interestingly, the oxidizing potential of Fe(CN)63- inside coacervates can be harnessed to drive the formation of new amide bonds between prebiotically relevant amino acids and α-amidothioacids. Aminoacylation is enhanced in Fe(CN)63-/peptide coacervates and selective for amino acids that interact less strongly with the coacervates. We finally use Fe(CN)63--containing coacervates to spatially control assembly of fibrous networks inside and at the surface of coacervate protocells. These results provide an important step towards the prebiotically relevant integration of redox chemistry in primitive cell-like compartments.

Advancing Biomimetic Functions of Synthetic Cells through Compartmentalized Cell-Free Protein Synthesis.

Synthetic cells are artificial constructs that mimic the structures and functions of living cells. They are attractive for studying diverse biochemical processes and elucidating the origins of life. While creating a living synthetic cell remains a grand challenge, researchers have successfully synthesized hundreds of unique synthetic cell platforms. One promising approach to developing more sophisticated synthetic cells is to integrate cell-free protein synthesis (CFPS) mechanisms into vesicle platforms. This makes it possible to create synthetic cells with complex biomimetic functions such as genetic circuits, autonomous membrane modifications, sensing and communication, and artificial organelles. This Review explores recent advances in the use of CFPS to impart advanced biomimetic structures and functions to bottom-up synthetic cell platforms. We also discuss the potential applications of synthetic cells in biomedicine as well as the future directions of synthetic cell research.

Construction of Membraneless and Multicompartmentalized Coacervate Protocells Controlling a Cell Metabolism-like Cascade Reaction.

In recent years, there has been growing attention to designing synthetic protocells, capable of mimicking micrometric and multicompartmental structures and highly complex physicochemical and biological processes with spatiotemporal control. Controlling metabolism-like cascade reactions in coacervate protocells is still challenging since signal transduction has to be involved in sequential and parallelized actions mediated by a pH change. Herein, we report the hierarchical construction of membraneless and multicompartmentalized protocells composed of (i) a cytosol-like scaffold based on complex coacervate droplets stable under flow conditions, (ii) enzyme-active artificial organelles and a substrate nanoreservoir capable of triggering a cascade reaction between them in response to a pH increase, and (iii) a signal transduction component based on the urease enzyme capable of the conversion of an exogenous biological fuel (urea) into an endogenous signal (ammonia and pH increase). Overall, this strategy allows a synergistic communication between their components within the membraneless and multicompartment protocells and, thus, metabolism-like enzymatic cascade reactions. This signal communication is transmitted through a scaffold protocell from an "inactive state" (nonfluorescent protocell) to an "active state" (fluorescent protocell capable of consuming stored metabolites).