Artificial Cell Engineering Research Articles

Designer peptide–DNA cytoskeletons regulate the function of synthetic cells

The bottom-up engineering of artificial cells requires a reconfigurable cytoskeleton that can organize at distinct locations and dynamically modulate its structural and mechanical properties. Here, inspired by the vast array of actin-binding proteins and their ability to reversibly crosslink or bundle filaments, we have designed a library of peptide–DNA crosslinkers varying in length, valency and geometry. Peptide filaments conjoint through DNA hybridization give rise to tactoid-shaped bundles with tunable aspect ratios and mechanics. When confined in cell-sized water-in-oil droplets, the DNA crosslinker design guides the localization of cytoskeletal structures at the cortex or within the lumen of the synthetic cells. The tunable spatial arrangement regulates the passive diffusion of payloads within the droplets and complementary DNA handles allow for the reversible recruitment and release of payloads on and off the cytoskeleton. Heat-induced reconfiguration of peptide–DNA architectures triggers shape deformations of droplets, regulated by DNA melting temperatures. Altogether, the modular design of peptide–DNA architectures is a powerful strategy towards the bottom-up assembly of synthetic cells.

Physical Concept to Explain the Regulation of Lipid Membrane Phase Separation under Isothermal Conditions.

Lateral phase separation within lipid bilayer membranes has attracted considerable attention in the fields of biophysics and cell biology. Living cells organize laterally segregated compartments, such as raft domains in an ordered phase, and regulate their dynamic structures under isothermal conditions to promote cellular functions. Model membrane systems with minimum components are powerful tools for investigating the basic phenomena of membrane phase separation. With the use of such model systems, several physicochemical characteristics of phase separation have been revealed. This review focuses on the isothermal triggering of membrane phase separation from a physical point of view. We consider the free energy of the membrane that describes lateral phase separation and explain the experimental results of model membranes to regulate domain formation under isothermal conditions. Three possible regulation factors are discussed: electrostatic interactions, chemical reactions and membrane tension. These findings may contribute to a better understanding of membrane lateral organization within living cells that function under isothermal conditions and could be useful for the development of artificial cell engineering.

Charge controlled interactions between DNA-modified silica nanoparticles and fluorosurfactants in microfluidic water-in-oil droplets.

Microfluidic droplets are an important tool for studying and mimicking biological systems, e.g., to examine with high throughput the interaction of biomolecular components and the functionality of natural cells, or to develop basic principles for the engineering of artificial cells. Of particular importance is the approach to generate a biomimetic membrane by supramolecular self-assembly of nanoparticle components dissolved in the aqueous phase of the droplets at the inner water/oil interface, which can serve both to mechanically reinforce the droplets and as an interaction surface for cells and other components. While this interfacial assembly driven by electrostatic interaction of surfactants is quite well developed for water/mineral oil (W/MO) systems, no approaches have yet been described to exploit this principle for water/fluorocarbon oil (W/FO) emulsion droplets. Since W/FO systems exhibit not only better compartmentalization but also gas solubility properties, which is particularly crucial for live cell encapsulation and cultivation, we report here the investigation of charged fluorosurfactants for the self-assembly of DNA-modified silica nanoparticles (SiNP-DNA) at the interface of microfluidic W/FO emulsions. To this end, an efficient multicomponent Ugi reaction was used to synthesize the novel fluorosurfactant M4SURF to study the segregation and accumulation of negatively charged SiNP-DNA at the inner interface of microfluidic droplets. Comparative measurements were performed with the negatively charged fluorosurfactant KRYTOX, which can also induce SiNP-DNA segregation in the presence of cations. The segregation dynamics is characterized and preliminary results of cell encapsulation in the SiNP-DNA functionalized droplets are shown.

Application of polymersomes in membrane protein study and drug discovery: Progress, strategies, and perspectives.

Membrane proteins (MPs) play key roles in cellular signaling pathways and are responsible for intercellular and intracellular interactions. Dysfunctional MPs are directly related to the pathogenesis of various diseases, and they have been exploited as one of the most sought-after targets in the pharmaceutical industry. However, working with MPs is difficult given that their amphiphilic nature requires protection from biological membrane or membrane mimetics. Polymersomes are bilayered nano-vesicles made of self-assembled block copolymers that have been widely used as cell membrane mimetics for MP reconstitution and in engineering of artificial cells. This review highlights the prevailing trend in the application of polymersomes in MP study and drug discovery. We begin with a review on the techniques for synthesis and characterization of polymersomes as well as methods of MP insertion to form proteopolymersomes. Next, we review the structural and functional analysis of the different types of MPs reconstituted in polymersomes, including membrane transport proteins, MP complexes, and membrane receptors. We then summarize the factors affecting reconstitution efficiency and the quality of reconstituted MPs for structural and functional studies. Additionally, we discuss the potential in using proteopolymersomes as platforms for high-throughput screening (HTS) in drug discovery to identify modulators of MPs. We conclude by providing future perspectives and recommendations on advancing the study of MPs and drug development using proteopolymersomes.

PH‐Gated Activation of Gene Transcription and Translation in Biocatalytic Metal–Organic Framework Artificial Cells

Regulating gene expression is an important goal for the development of artificial cells that have the ability to replicate the functions of living cells. Despite that, it remains a critical challenge to the engineering of artificial cells that can self‐regulate genetic expression in response to the “extracellular” environments. Herein, the gene transcription and translation engineered into metal–organic framework (MOF)‐based artificial cells using extracellular pH as a versatile gate to control green fluorescence protein (GFP) expression is reported for the first time. The results show that the artificial cells can metabolize biological molecules through an engineered multistep process of biocatalytic reactions, and importantly, sense acidic pH in the extracellular environment to initiate gene expression, mimicking natural cells. Moreover, co‐culturing with living cells further prove the adaptive cellular responses in the artificial cells when the target proteins can be produced in response to the change in local cell environments. The versatility of the gene expression regulation mechanism and the sensing of external stimuli by artificial cells supports the goal to bring protocells one step closer to native cells and suggests the potential of the artificial systems in biosensing and therapy applications.

A platform for light-controlled formation of free-stranding lipid membranes

The engineering of artificial cells is one of the most significant scientific challenges. Thus, controlled fabrication and in situ monitoring of biomimetic nanoscale objects are among the central issues in current science and technology. Studies of transmembrane channels and cell mechanics often require the formation of lipid bilayers (LBs), their modification and their transfer to a particular place. We present here a novel approach for remotely controlled manipulation of LBs. Layer-by-layer deposition of polyethyleneimine and poly(sodium 4-styrenesulfonate) on a nanostructured TiO 2 photoanode was performed to obtain a surface with the desired net charge and to enhance photocatalytic performance. The LB was deposited on top of a multi-layer positive polymer cushion by the dispersion of negative vesicles. The separation distance between the electrostatically linked polyelectrolyte cushion and the LB can be adjusted by changing the environmental pH, as zwitter-ionic lipid molecules undergo pH-triggered charge-shifting. Protons were generated remotely by photoanodic water decomposition on the TiO 2 surface under 365 nm illumination. The resulting pH gradient was characterized by scanning vibrating electrode and scanning ion-selective electrode techniques. The light-induced reversible detachment of the LB from the polymer-cushioned photoactive substrate was found to correlate with suggested impedance models.

Stimuli-Triggered Activity of Nanoreactors by Biomimetic Engineering Polymer Membranes.

The development of advanced stimuli-responsive systems for medicine, catalysis, or technology requires compartmentalized reaction spaces with triggered activity. Only very few stimuli-responsive systems preserve the compartment architecture, and none allows a triggered activity in situ. We present here a biomimetic strategy to molecular transmembrane transport by engineering synthetic membranes equipped with channel proteins so that they are stimuli-responsive. Nanoreactors with triggered activity were designed by simultaneously encapsulating an enzyme inside polymer compartments, and inserting protein "gates" in the membrane. The outer membrane protein F (OmpF) porin was chemically modified with a pH-responsive molecular cap to serve as "gate" producing pH-driven molecular flow through the membrane and control the in situ enzymatic activity. This strategy provides complex reaction spaces necessary in "smart" medicine and for biomimetic engineering of artificial cells.

Bioinspirations: Cell-Inspired Small-Scale Systems for Enabling Studies in Experimental Biomechanics

Biomechanical forces govern the behaviors of organisms and their environment and examining these behaviors to understand the underlying phenomena is an important challenge. One experimental approach for probing these interactions between organisms and their biomechanical environment uses biologically-inspired, artificial surrogates that reproduce organic mechanical systems. For the case of complex, multicellular organisms, robot surrogates have been particularly effective, such as in the analysis of the fins of fish and insects' wings. This biologically-inspired approach is also exciting when examining cell-scale responses as multicellular organisms' behavior is directly influenced by the integrated interactions of smaller-scale components (i.e., cells). In this review, we introduce the burgeoning field of engineering of artificial cells, which focuses on developing cell-scale entities replicating cellular behaviors. We describe both a bottom-up approach to constructing artificial cells, using molecular components to directly assemble artificial cells, as well as a top-down approach, in which living cells are encapsulated in a single entity whose behavior is determined by its constituent members. In particular, we discuss the potential role of these artificial cells as implantable controllers, designed to alter the mechanical behavior of a host organism. Eventually, artificial cells designed to function as small-scale controllers may help alter organisms' phenotypes.

Synthesis and utilization of E. coli‐encapsulated PEG‐based microdroplet using a microfluidic chip for biological application

We report herein an effective strategy for encapsulating Escherichia coli in polyethylene glycol diacrylate (PEGDA) microdroplets using a microfluidic device and chemical polymerization. PEGDA was employed as a reactant due to the biocompatibility, high porosity, and hydrophilic property. The uniform size and shape of microdroplets are obtained in a single-step process using microfluidic device. The size of microdroplets can be controlled through the changing continuous flow rate. The combination of microdroplet generation and chemical polymerization techniques provide unique environment to produce non-toxic ways of fabricating microorganism-encapsulated hydrogel microbeads. Due to these unique properties of micro-sized hydrogel microbeads, the encapsulated E. coli can maintain viability inside of microbeads and green fluorescent protein (GFP) and red fluorescent protein (RFP) genes are efficiently expressed inside of microbeads after isopropyl-β-D-thiogalactopyranoside induction, suggesting that there is no low-molecular weight substrate transfer limitation inside of microbeads. Furthermore, non-toxic, gentle, and outstanding biocompatibility of microbeads, the encapsulated E. coli can be used in various applications including biotransformation, biosensing, bioremediation, and engineering of artificial cells.