Programmable Enzymatic Reaction Network in Artificial Cell-Like Polymersomes.

The ability to precisely control in vitro enzymatic reactions in synthetic cells plays a crucial role in the bottom-up design of artificial cell models that can recapitulate the key cellular features and functions such as metabolism. However, integration of enzymatic reactions has been limited to bulk or microfluidic emulsions without a membrane, lacking the ability to design more sophisticated higher-order artificial cell communities for reconstituting spatiotemporal biological information at multiple length scales. Herein, droplet microfluidics is utilized to synthesize artificial cell-like polymersomes with distinct molecular permeability for spatiotemporal control of enzymatic reactions driven by external signals and fuels. The presence of a competing reverse enzymatic reaction that depletes the active substrates is shown to enable demonstration of fuel-driven formation of sub-microcompartments within polymersomes as well as realization of out-of-equilibrium systems. In addition, the different permeability characteristics of polymersome membranes are exploited to successfully construct a programmable enzymatic reaction network that mimics cellular communication within a heterogeneous cell community through selective molecular transport.

High-Precision Synthesis of RNA-Loaded Lipid Nanoparticles for Biomedical Applications.

The recent development of RNA-based therapeutics in delivering nucleic acids for gene editing and regulating protein translation has led to the effective treatment of various diseases including cancer, inflammatory and genetic disorder, as well as infectious diseases. Among these, lipid nanoparticles (LNP) have emerged as a promising platform for RNA delivery and have shed light by resolving the inherent instability issues of naked RNA and thereby enhancing the therapeutic potency. These LNP consisting of ionizable lipid, helper lipid, cholesterol, and poly(ethylene glycol)-anchored lipid can stably enclose RNA and help them release into the cells' cytosol. Herein, the significant progress made in LNP research starting from the LNP constituents, formulation, and their diverse applications is summarized first. Moreover, the microfluidic methodologies which allow precise assembly of these newly developed constituents to achieve LNP with controllable composition and size, high encapsulation efficiency as well as scalable production are highlighted. Furthermore, a short discussion on current challenges as well as an outlook will be given on emerging approaches to resolving these issues.

Spatiotemporal control of signal-driven enzymatic reaction in artificial cell-like polymersomes.

Living cells can spatiotemporally control biochemical reactions to dynamically assemble membraneless organelles and remodel cytoskeleton. Herein, we present a microfluidic approach to prepare semi-permeable polymersomes comprising of amphiphilic triblock copolymer to achieve external signal-driven complex coacervation as well as biophysical reconstitution of cytoskeleton within the polymersomes. We also show that the microfluidic synthesis of polymersomes enables precise control over size, efficient encapsulation of enzymes as well as regulation of substrates without the use of biopores. Moreover, we demonstrate that the resulting triblock copolymer-based membrane in polymersomes is size-selective, allowing phosphoenol pyruvate to readily diffuse through the membrane and induce enzymatic reaction and successive coacervation or actin polymerization in the presence of pyruvate kinase and adenosine diphosphate inside the polymersomes. We envision that the Pluronic-based polymersomes presented in this work will shed light in the design of in vitro enzymatic reactions in artificial cell-like vesicles.

Recent developments in microfluidic synthesis of artificial cell-like polymersomes and liposomes for functional bioreactors.

Recent advances in droplet microfluidics have led to the fabrication of versatile vesicles with a structure that mimics the cellular membrane. These artificial cell-like vesicles including polymersomes and liposomes effectively enclose an aqueous core with well-defined size and composition from the surrounding environment to implement various biological reactions, serving as a diverse functional reactor. The advantage of realizing various biological phenomena within a compartment separated by a membrane that resembles a natural cell membrane is actively explored in the fields of synthetic biology as well as biomedical applications including drug delivery, biosensors, and bioreactors, to name a few. In this Perspective, we first summarize various methods utilized in producing these polymersomes and liposomes. Moreover, we will highlight some of the recent advances in the design of these artificial cell-like vesicles for functional bioreactors and discuss the current issues and future perspectives.

Aqueous Two-Phase System (ATPS)-Based Polymersomes for Particle Isolation and Separation.

Aqueous two-phase systems (ATPSs) have been widely used in the separation, purification, and enrichment of biomolecules for their excellent biocompatibility. While ultracentrifugation and microfluidic devices have been combined with ATPS to facilitate the separation of biomolecules and achieve high recovery yields, they often lack the ability to effectively isolate and separate biomolecules in low concentrations. In this work, we present a strategy that leverages the preferential partitioning of biomolecules in ATPS droplets to efficiently separate model extracellular vesicle (EV) particles. We demonstrate that the additional oil phase between the inner ATPS droplets and the aqueous continuous phase in triple emulsion droplets resolves the size controllability and instability issues of ATPS droplets, enabling the production of highly monodisperse ATPS-based polymersomes with enhanced stability for effective isolation of ATPS droplets from the surrounding environment. Furthermore, we achieve separation of model EV particles in a single dextran (DEX)-rich droplet by the massive production of ATPS-based polymersomes and osmotic-pressure-induced rupture of the selected polymersome in a hypertonic solution composed of poly(ethylene glycol) (PEG).