Genetically programmed synthetic cells for thermo-responsive protein synthesis and cargo release.

Synthetic cells containing genetic programs and protein expression machinery are increasingly recognized as powerful counterparts to engineered living cells in the context of biotechnology, therapeutics and cellular modelling. So far, genetic regulation of synthetic cell activity has been largely confined to chemical stimuli; to unlock their potential in applied settings, engineering stimuli-responsive synthetic cells under genetic regulation is imperative. Here we report the development of temperature-sensitive synthetic cells that control protein production by exploiting heat-responsive mRNA elements. This is achieved by combining RNA thermometer technology, cell-free protein expression and vesicle-based synthetic cell design to create cell-sized capsules able to initiate synthesis of both soluble proteins and membrane proteins at defined temperatures. We show that the latter allows for temperature-controlled cargo release phenomena with potential implications for biomedicine. Platforms like the one presented here can pave the way for customizable, genetically programmed synthetic cells under thermal control to be used in biotechnology.

Biomimetic behaviors in hydrogel artificial cells through embedded organelles.

Artificial cells are biomimetic structures formed from molecular building blocks that replicate biological processes, behaviors, and architectures. Of these building blocks, hydrogels have emerged as ideal, yet underutilized candidates to provide a gel-like chassis in which to incorporate both biological and nonbiological componentry which enables the replication of cellular functionality. Here, we demonstrate a microfluidic strategy to assemble biocompatible cell-sized hydrogel-based artificial cells with a variety of different embedded functional subcompartments, which act as engineered synthetic organelles. The organelles enable the recreation of increasingly biomimetic behaviors, including stimulus-induced motility, content release through activation of membrane-associated proteins, and enzymatic communication with surrounding bioinspired compartments. In this way, we showcase a foundational strategy for the bottom-up construction of hydrogel-based artificial cell microsystems which replicate fundamental cellular behaviors, paving the way for the construction of next-generation biotechnological devices.

Stimuli-responsive vesicles as distributed artificial organelles for bacterial activation.

Intercellular communication is a hallmark of living systems. As such, engineering artificial cells that possess this behavior has been at the heart of activities in bottom-up synthetic biology. Communication between artificial and living cells has potential to confer novel capabilities to living organisms that could be exploited in biomedicine and biotechnology. However, most current approaches rely on the exchange of chemical signals that cannot be externally controlled. Here, we report two types of remote-controlled vesicle-based artificial organelles that translate physical inputs into chemical messages that lead to bacterial activation. Upon light or temperature stimulation, artificial cell membranes are activated, releasing signaling molecules that induce protein expression in Escherichia coli. This distributed approach differs from established methods for engineering stimuli-responsive bacteria. Here, artificial cells (as opposed to bacterial cells themselves) are the design unit. Having stimuli-responsive elements compartmentalized in artificial cells has potential applications in therapeutics, tissue engineering, and bioremediation. It will underpin the design of hybrid living/nonliving systems where temporal control over population interactions can be exerted.

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.

Modular Bioorthogonal Lipid Nanoparticle Modification Platforms for Cardiac Homing.

Lipid nanoparticles (LNPs) are becoming widely adopted as vectors for the delivery of therapeutic payloads but generally lack intrinsic tissue-homing properties. These extracellular vesicle (EV) mimetics can be targeted toward the liver, lung, or spleen via charge modification of their lipid headgroups. Homing to other tissues has only been achieved via covalent surface modification strategies using small-molecule ligands, peptides, or monoclonal antibodies─methods that are challenging to couple with large-scale manufacturing. Herein, we design a novel modular artificial membrane-binding protein (AMBP) platform for the modification of LNPs postformation. The system is composed of two protein modules that can be readily coupled using bioorthogonal chemistry to yield the AMBP. The first is a membrane anchor module comprising a supercharged green fluorescent protein (scGFP) electrostatically conjugated to a dynamic polymer surfactant corona. The second is a functional module containing a cardiac tissue fibronectin homing sequence from the bacterial adhesin CshA. We demonstrate that LNPs modified using the AMBP exhibit a 20-fold increase in uptake by fibronectin-rich C2C12 cells under static conditions and a 10-fold increase under physiologically relevant shear stresses, with no loss of cell viability. Moreover, we show targeted localization of the AMBP-modified LNPs in zebrafish hearts, highlighting their therapeutic potential as a vector for the treatment of cardiac disease and, more generally, as a smart vector.

Building a synthetic mechanosensitive signaling pathway in compartmentalized artificial cells.

To date, reconstitution of one of the fundamental methods of cell communication, the signaling pathway, has been unaddressed in the bottom-up construction of artificial cells (ACs). Such developments are needed to increase the functionality and biomimicry of ACs, accelerating their translation and application in biotechnology. Here, we report the construction of a de novo synthetic signaling pathway in microscale nested vesicles. Vesicle-cell models respond to external calcium signals through activation of an intracellular interaction between phospholipase A2 and a mechanosensitive channel present in the internal membranes, triggering content mixing between compartments and controlling cell fluorescence. Emulsion-based approaches to AC construction are therefore shown to be ideal for the quick design and testing of new signaling networks and can readily include synthetic molecules difficult to introduce to biological cells. This work represents a foundation for the engineering of multicompartment-spanning designer pathways that can be utilized to control downstream events inside an AC, leading to the assembly of micromachines capable of sensing and responding to changes in their local environment.

Acylation of the Incretin Peptide Exendin-4 Directly Impacts Glucagon-Like Peptide-1 Receptor Signaling and Trafficking.

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor and mainstay therapeutic target for the treatment of type 2 diabetes and obesity. Recent reports have highlighted how biased agonism at the GLP-1R affects sustained glucose-stimulated insulin secretion through avoidance of desensitization and downregulation. A number of GLP-1R agonists (GLP-1RAs) feature a fatty acid moiety to prolong their pharmacokinetics via increased albumin binding, but the potential for these chemical changes to influence GLP-1R function has rarely been investigated beyond potency assessments for cAMP. Here, we directly compare the prototypical GLP-1RA exendin-4 with its C-terminally acylated analog, exendin-4-C16. We examine relative propensities of each ligand to recruit and activate G proteins and ß-arrestins, endocytic and postendocytic trafficking profiles, and interactions with model and cellular membranes in HEK293 and HEK293T cells. Both ligands had similar cAMP potency, but exendin-4-C16 showed ∼2.5-fold bias toward G protein recruitment and a ∼60% reduction in ß-arrestin-2 recruitment efficacy compared with exendin-4, as well as reduced GLP-1R endocytosis and preferential targeting toward recycling pathways. These effects were associated with reduced movement of the GLP-1R extracellular domain measured using a conformational biosensor approach and a ∼70% increase in insulin secretion in INS-1 832/3 cells. Interactions with plasma membrane lipids were enhanced by the acyl chain. Exendin-4-C16 showed extensive albumin binding and was highly effective for lowering of blood glucose in mice over at least 72 hours. Our study highlights the importance of a broad approach to the evaluation of GLP-1RA pharmacology. SIGNIFICANCE STATEMENT: Acylation is a common strategy to enhance the pharmacokinetics of peptide-based drugs. This work shows how acylation can also affect various other pharmacological parameters, including biased agonism, receptor trafficking, and interactions with the plasma membrane, which may be therapeutically important.

Manufacture of Multilayered Artificial Cell Membranes through Sequential Bilayer Deposition on Emulsion Templates.

Efforts to manufacture artificial cells that replicate the architectures, processes and behaviours of biological cells are rapidly increasing. Perhaps the most commonly reconstructed cellular structure is the membrane, through the use of unilamellar vesicles as models. However, many cellular membranes, including bacterial double membranes, nuclear envelopes, and organelle membranes, are multilamellar. Due to a lack of technologies available for their controlled construction, multilayered membranes are not part of the repertoire of cell-mimetic motifs used in bottom-up synthetic biology. To address this, we developed emulsion-based technologies that allow cell-sized multilayered vesicles to be produced layer-by-layer, with compositional control over each layer, thus enabling studies that would otherwise remain inaccessible. We discovered that bending rigidities scale with the number of layers and demonstrate inter-bilayer registration between coexisting liquid-liquid domains. These technologies will contribute to the exploitation of multilayered membrane structures, paving the way for incorporating protein complexes that span multiple bilayers.

The effect of headgroup methylation on polymorphic phase behaviour in hydrated N-methylated phosphoethanolamine:palmitic acid membranes.

Mixtures of fatty acids and phospholipids can form hexagonal (HII) and inverse bicontinuous cubic phases, the latter of which are implicated in various cellular processes and have wide-ranging biotechnological applications in protein crystallisation and drug delivery systems. Therefore, it is vitally important to understand the formation conditions of inverse bicontinuous cubic phases and how their properties can be tuned. We have used differential scanning calorimetry and synchrotron-based small angle and wide angle X-ray scattering (SAXS/WAXS) to investigate the polymorphic phase behaviour of palmitic acid/partially-methylated phospholipid mixtures, and how headgroup methylation impacts on inverse bicontinuous cubic phase formation. We find that upon partial methylation of the phospholipid headgroup (1 or 2 methyl substituents) inverse bicontinuous cubic phases are formed (of the Im3m spacegroup), which is not the case with 0 or 3 methyl substituents. This shows how important headgroup methylation is for controlling phase behaviour and how a change in headgroup methylation can be used to controllably tune various inverse bicontinuous phase features such as their lattice parameter and the temperature range of their stability.

Microfluidic technologies for the synthesis and manipulation of biomimetic membranous nano-assemblies.

Microfluidics has been proposed as an attractive alternative to conventional bulk methods used in the generation of self-assembled biomimetic structures, particularly where there is a desire for more scalable production. The approach also allows for greater control over the self-assembly process, and parameters such as particle architecture, size, and composition can be finely tuned. Microfluidic techniques used in the generation of microscale assemblies (giant vesicles and higher-order multi-compartment assemblies) are fairly well established. These tend to rely on microdroplet templation, and the resulting structures have found use as comparmentalised motifs in artificial cells. Challenges in generating sub-micron droplets have meant that reconfiguring this approach to form nano-scale structures is not straightforward. This is beginning to change however, and recent technological advances have instigated the manufacture and manipulation of an increasingly diverse repertoire of biomimetic nano-assemblies, including liposomes, polymersomes, hybrid particles, multi-lamellar structures, cubosomes, hexosomes, nanodiscs, and virus-like particles. The following review will discuss these higher-order self-assembled nanostructures, including their biochemical and industrial applications, and techniques used in their production and analysis. We suggest ways in which existing technologies could be repurposed for the enhanced design, manufacture, and exploitation of these structures and discuss potential challenges and future research directions. By compiling recent advances in this area, it is hoped we will inspire future efforts toward establishing scalable microfluidic platforms for the generation of biomimetic nanoparticles of enhanced architectural and functional complexity.

Interfacing Living and Synthetic Cells as an Emerging Frontier in Synthetic Biology.

The construction of artificial cells from inanimate molecular building blocks is one of the grand challenges of our time. In addition to being used as simplified cell models to decipher the rules of life, artificial cells have the potential to be designed as micromachines deployed in a host of clinical and industrial applications. The attractions of engineering artificial cells from scratch, as opposed to re-engineering living biological cells, are varied. However, it is clear that artificial cells cannot currently match the power and behavioural sophistication of their biological counterparts. Given this, many in the synthetic biology community have started to ask: is it possible to interface biological and artificial cells together to create hybrid living/synthetic systems that leverage the advantages of both? This article will discuss the motivation behind this cellular bionics approach, in which the boundaries between living and non-living matter are blurred by bridging top-down and bottom-up synthetic biology. It details the state of play of this nascent field and introduces three generalised hybridisation modes that have emerged.

New Directions for Artificial Cells Using Prototyped Biosystems.

Microfluidics has has enabled the generation of  a range of single compartment and multicompartment vesicles and bilayer-delineated droplets that can be assembled in 2D and 3D. These model systems are becoming increasingly used as artificial cell chassis and as biomimetic constructs for assembling tissue models, engineering therapeutic delivery systems, and screening drugs. One bottleneck in developing this technology is the time, expertise, and equipment required for device fabrication. This has led to interest across the microfluidics community in using rapid prototyping to engineer microfluidic devices from computer-aided-design (CAD) drawings. We highlight how this rapid-prototyping revolution is transforming the fabrication of microfluidic devices for artificial cell construction in bottom-up synthetic biology. We provide an outline of the current landscape and present how advances in the field may give rise to the next generation of multifunctional biodevices, particularly with Industry 4.0 on the horizon. Successfully developing this technology and making it open-source could pave the way for a new generation of citizen-led science, fueling the possibility that the next multibillion-dollar start-up could emerge from an attic or a basement.

Construction of membrane-bound artificial cells using microfluidics: a new frontier in bottom-up synthetic biology.

The quest to construct artificial cells from the bottom-up using simple building blocks has received much attention over recent decades and is one of the grand challenges in synthetic biology. Cell mimics that are encapsulated by lipid membranes are a particularly powerful class of artificial cells due to their biocompatibility and the ability to reconstitute biological machinery within them. One of the key obstacles in the field centres on the following: how can membrane-based artificial cells be generated in a controlled way and in high-throughput? In particular, how can they be constructed to have precisely defined parameters including size, biomolecular composition and spatial organization? Microfluidic generation strategies have proved instrumental in addressing these questions. This article will outline some of the major principles underpinning membrane-based artificial cells and their construction using microfluidics, and will detail some recent landmarks that have been achieved.

Optically assembled droplet interface bilayer (OptiDIB) networks from cell-sized microdroplets.

We report a new platform technology to systematically assemble droplet interface bilayer (DIB) networks in user-defined 3D architectures from cell-sized droplets using optical tweezers. Our OptiDIB platform is the first demonstration of optical trapping to precisely construct 3D DIB networks, paving the way for the development of a new generation of modular bio-systems.

Protein synthesis in artificial cells: using compartmentalisation for spatial organisation in vesicle bioreactors.

Whereas spatial organisation of function is ubiquitous in biology, it has been lacking in artificial cells. We rectify this by using multi-compartment vesicles as chassis for artificial cells, allowing distinct biological processes to be isolated in space. This is demonstrated by in vitro synthesis of two proteins in predefined vesicle regions.

Visualizing Actin Packing and the Effects of Actin Attachment on Lipid Membrane Viscosity Using Molecular Rotors.

The actin cytoskeleton and its elaborate interplay with the plasma membrane participate in and control numerous biological processes in eukaryotic cells. Malfunction of actin networks and changes in their dynamics are related to various diseases, from actin myopathies to uncontrolled cell growth and tumorigenesis. Importantly, the biophysical and mechanical properties of actin and its assemblies are deeply intertwined with the biological functions of the cytoskeleton. Novel tools to study actin and its associated biophysical features are, therefore, of prime importance. Here we develop a new approach which exploits fluorescence lifetime imaging microscopy (FLIM) and environmentally sensitive fluorophores termed molecular rotors, acting as quantitative microviscosity sensors, to monitor dynamic viscoelastic properties of both actin structures and lipid membranes. In order to reproduce a minimal actin cortex in synthetic cell models, we encapsulated and attached actin networks to the lipid bilayer of giant unilamellar vesicles (GUVs). Using a cyanine-based molecular rotor, DiSC2(3), we show that different types of actin bundles are characterized by distinct packing, which can be spatially resolved using FLIM. Similarly, we show that a lipid bilayer-localized molecular rotor can monitor the effects of attaching cross-linked actin networks to the lipid membrane, revealing an increase in membrane viscosity upon actin attachment. Our approach bypasses constraints associated with existing methods for actin imaging, many of which lack the capability for direct visualization of biophysical properties. It can therefore contribute to a deeper understanding of the role that actin plays in fundamental biological processes and help elucidate the underlying biophysics of actin-related diseases.

Engineered Bacteria as Living Biosensors in Dermal Tattoos.

Dermal tattoo biosensors are promising platforms for real-time monitoring of biomarkers, with skin used as a diagnostic interface. Traditional tattoo sensors have utilized small molecules as biosensing elements. However, the rise of synthetic biology has enabled the potential employment of engineered bacteria as living analytical tools. Exploiting engineered bacterial sensors will allow for potentially more sensitive detection across a broad biomarker range, with advanced processing and sense/response functionalities using genetic circuits. Here, the interfacing of bacterial biosensors as living analytics in tattoos is shown. Engineered bacteria are encapsulated into micron-scale hydrogel beads prepared through scalable microfluidics. These biosensors can sense both biochemical cues (model biomarkers) and biophysical cues (temperature changes, using RNA thermometers), with fluorescent readouts. By tattooing beads into skin models and confirming sensor activity post-tattooing, our study establishes a foundation for integrating bacteria as living biosensing entities in tattoos.

Engineering a nanoscale liposome-in-liposome for in situ biochemical synthesis and multi-stage release.

Soft-matter nanoscale assemblies such as liposomes and lipid nanoparticles have the potential to deliver and release multiple cargos in an externally stimulated and site-specific manner. Such assemblies are currently structurally simplistic, comprising spherical capsules or lipid clusters. Given that form and function are intertwined, this lack of architectural complexity restricts the development of more sophisticated properties. To address this, we have devised an engineering strategy combining microfluidics and conjugation chemistry to synthesize nanosized liposomes with two discrete compartments, one within another, which we term concentrisomes. We can control the composition of each bilayer and tune both particle size and the dimensions between inner and outer membranes. We can specify the identity of encapsulated cargo within each compartment, and the biophysical features of inner and outer bilayers, allowing us to imbue each bilayer with different stimuli-responsive properties. We use these particles for multi-stage release of two payloads at defined time points, and as attolitre reactors for triggered in situ biochemical synthesis.

Lipid vesicle-based molecular robots.

A molecular robot, which is a system comprised of one or more molecular machines and computers, can execute sophisticated tasks in many fields that span from nanomedicine to green nanotechnology. The core parts of molecular robots are fairly consistent from system to system and always include (i) a body to encapsulate molecular machines, (ii) sensors to capture signals, (iii) computers to make decisions, and (iv) actuators to perform tasks. This review aims to provide an overview of approaches and considerations to develop molecular robots. We first introduce the basic technologies required for constructing the core parts of molecular robots, describe the recent progress towards achieving higher functionality, and subsequently discuss the current challenges and outlook. We also highlight the applications of molecular robots in sensing biomarkers, signal communications with living cells, and conversion of energy. Although molecular robots are still in their infancy, they will unquestionably initiate massive change in biomedical and environmental technology in the not too distant future.