Controlled Membrane Transport in Polymeric Biomimetic Nanoreactors.

Polymersome-based biomimetic nanoreactors (PBNs) have generated great interest in nanomedicine and cell mimicry due to their robustness, tuneable chemistry, and broad applicability in biologically relevant fields. In this concept review, we mainly discuss the state of the art in functional polymersomes as biomimetic nanoreactors with membrane-controlled transport. PBNs that use environmental changes or external stimuli to adjust membrane permeability while maintaining structural integrity are highlighted. By encapsulating catalytic species, PBNs are able to convert inactive substrates into functional products in a controlled manner. In addition, special attention is paid to the use of PBNs as tailored artificial organelles with biomedical applications in vitro and in vivo, facilitating the fabrication of next-generation artificial organelles as therapeutic nanocompartments.

Confining the Sol-Gel Reaction at the Water/Oil Interface: Creating Compartmentalized Enzymatic Nano-Organelles for Artificial Cells.

Living organisms compartmentalize their catalytic reactions in membranes for increased efficiency and selectivity. To mimic the organelles of eukaryotic cells, we develop a mild approach for in situ encapsulating enzymes in aqueous-core silica nanocapsules. In order to confine the sol-gel reaction at the water/oil interface of miniemulsion, we introduce an aminosilane to the silica precursors, which serves as both catalyst and an amphiphilic anchor that electrostatically assembles with negatively charged hydrolyzed alkoxysilanes at the interface. The semi-permeable shell protects enzymes from proteolytic attack, and allows the transport of reactants and products. The enzyme-carrying nanocapsules, as synthetic nano-organelles, are able to perform cascade reactions when enveloped in a polymer vesicle, mimicking the hierarchically compartmentalized reactions in eukaryotic cells. This in situ encapsulation approach provides a versatile platform for the delivery of biomacromolecules.

Synthetic Silica Nano-Organelles for Regulation of Cascade Reactions in Multi-Compartmentalized Systems.

In eukaryotic cells, enzymes are compartmentalized into specific organelles so that different reactions and processes can be performed efficiently and with a high degree of control. In this work, we show that these features can be artificially emulated in robust synthetic organelles constructed using an enzyme co-compartmentalization strategy. We describe an in situ encapsulation approach that allows enzymes to be loaded into silica nanoreactors in well-defined compositions. The nanoreactors can be combined into integrated systems to produce a desired reaction outcome. We used the selective enzyme co-compartmentalization and nanoreactor integration to regulate competitive cascade reactions and to modulate the kinetics of sequential reactions involving multiple nanoreactors. Furthermore, we show that the nanoreactors can be efficiently loaded into giant polymer vesicles, resulting in multi-compartmentalized microreactors.

Membrane Manipulation of Giant Unilamellar Polymer Vesicles with a Temperature-Responsive Polymer.

Understanding the complex behavior and dynamics of cellular membranes is integral to gain insight into cellular division and fusion processes. Bottom-up synthetic cells are as a platform for replicating and probing cellular behavior. Giant polymer vesicles are more robust than liposomal counterparts, as well as having a broad range of chemical functionalities. However, the stability of the membrane can prohibit dynamic processes such as membrane phase separation and division. Here, we present a method for manipulating the membrane of giant polymersomes using a temperature responsive polymer. Upon elevation of temperature deformation and phase separation of the membrane was observed. Upon cooling, the membrane relaxed and became homogeneous again, with infrequent division of the synthetic cells.

Formation of giant polymer vesicles by simple double emulsification using block copolymers as the sole surfactant.

Polymer vesicles that mimic the function of cell membranes can be obtained through the self-assembly of amphiphilic block copolymers. The cell-like characteristics of polymer vesicles, such as the core-shell structure, semi-permeability and tunable surface chemistry make them excellent building blocks for artificial cells. However, the standard preparation methods for polymer vesicles can be time consuming, require special equipment, or have low encapsulation efficiency for large components, such as nanomaterials and proteins. Here, we introduce a new encapsulation strategy based on a simple double emulsification (SDE) approach which allows giant polymer vesicles to be formed in a short time and with basic laboratory equipment. The SDE method requires a single low molecular weight block copolymer that has the dual role of macromolecular surfactant and membrane building block. Giant polymer vesicles with diameters between 20-50 µm were produced, which allowed proteins and nanoparticles to be encapsulated. To demonstrate its practical application, we used the SDE method to assemble a simple artificial cell that mimics a two-step enzymatic cascade reaction. The SDE method described here introduces a new tool for simple and rapid fabrication of synthetic compartments.

Polymer-Based Module for NAD+ Regeneration with Visible Light.

The regeneration of enzymatic cofactors by cell-free synthetic modules is a key step towards producing a purely synthetic cell. Herein, we demonstrate the regeneration of the enzyme cofactor NAD+ by photo-oxidation of NADH under visible-light irradiation by using metal-free conjugated polymer nanoparticles. Encapsulation of the light-active nanoparticles in the lumen of polymeric vesicles produced a fully organic module able to regenerate NAD+ in an enzyme-free system. The polymer compartment conferred physical and chemical autonomy to the module, allowing the regeneration of NAD+ to occur efficiently, even in harsh chemical environments. Moreover, we show that regeneration of NAD+ by the photocatalyst nanoparticles can oxidize a model substrate, in conjunction with the enzyme glycerol dehydrogenase. To ensure the longevity of the enzyme, we immobilized it within a protective silica matrix; this yielded enzymatic silica nanoparticles with enhanced long-term performance and compatibility with the NAD+ -regeneration system.

Self-Assembly of Giant Polymer Vesicles by Light-Assisted Solid Hydration.

An amphiphilic copolymer containing a terminal spiropyran (P-SP) forms giant polymer vesicles when exposed to ultraviolet (UV) light. The process involves photoisomerization of the spiropyran to the corresponding hydrophilic merocyanine isomer, which significantly improves the efficiency of film hydration and facilitates the self-assembly of the polymer in water. Giant vesicles formed by light-assisted hydration have diameters ranging from 5 to 25 micrometers, and can be observed and quantified by confocal fluorescence microscopy. Rapid and efficient formation of giant vesicles only occur during exposure of P-SP to UV light and within the area delimited by the light beam. Light-assisted hydration offers high spatial and temporal control over vesicle formation, conditions not easily fulfilled by other techniques.

A Reversible Proton Generator with On/Off Thermoswitch.

A reversible polymer photoacid with a thermal on/off switch at physiological temperature able to trigger a large pH modulation of its environment is prepared. Light is used to control the acidity of the solution. Additionally, the temperature could be used to modulate the photoacid efficiency, practically turning on and off the ability of the polymer to produce protons. The behavior of this thermoresponsive photoacid copolymer is the result of the combined action of the temperature-responsive N-isopropylacrylamide and of a reversible photoacid monomer based on a spiropyran derivative. The acidification of the aqueous medium is activated by irradiation at λ = 460 nm. The reverse reaction is achieved by removing the light stimuli or by exposing the solution to UV-light. Increasing the temperature above the lower critical solution temperature of the copolymer deactivates the photoacid and irradiation at λ = 460 nm does not lead to the generation of protons or to any detectable change in the pH value of the solution. Hence, the addition of N-isopropylacrylamide as a comonomer acts as a thermal on/off switch for the photoacid and the coupling of temperature-and light-responsiveness in the polyphotoacids yields a "thermophotoacid".

Asymmetric Covalent Triazine Framework for Enhanced Visible-Light Photoredox Catalysis via Energy Transfer Cascade.

Complex multiple-component semiconductor photocatalysts can be constructed that display enhanced catalytic efficiency via multiple charge and energy transfer, mimicking photosystems in nature. In contrast, the efficiency of single-component semiconductor photocatalysts is usually limited due to the fast recombination of the photogenerated excitons. Here, we report the design of an asymmetric covalent triazine framework as an efficient organic single-component semiconductor photocatalyst. Four different molecular donor-acceptor domains are obtained within the network, leading to enhanced photogenerated charge separation via an intramolecular energy transfer cascade. The photocatalytic efficiency of the asymmetric covalent triazine framework is superior to that of its symmetric counterparts; this was demonstrated by the visible-light-driven formation of benzophosphole oxides from diphenylphosphine oxide and diphenylacetylene.

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.

Assembly of biomimetic microreactors using caged-coacervate droplets.

Complex coacervates are liquid-like droplets that can be used to create adaptive cell-like compartments. These compartments offer a versatile platform for the construction of bioreactors inspired by living cells. However, the lack of a membrane significantly reduces the colloidal stability of coacervates in terms of fusion and surface wetting, which limits their suitability as compartments. Here, we describe the formation of caged-coacervates surrounded by a semipermeable shell of silica nanocapsules. We demonstrate that the silica nanocapsules create a protective shell that also regulates the molecular transport of water-soluble compounds as a function of nanocapasule size. The adjustable semipermeability and intrinsic affinity of enzymes for the interior of the caged-coacervates allowed us to assemble biomimetic microreactors with enhanced colloidal stability.

Polymeric Microreactors with pH-Controlled Spatial Localization of Cascade Reactions.

Lipid and polymer vesicles provide versatile means of creating systems that mimic the architecture of cells. However, these constructs cannot mimic the adaptive compartmentalization observed in cells, where the assembly and disassembly of subcompartments are dynamically modulated by environmental cues. Here, we describe a fully polymeric microreactor with a coacervate-in-vesicle architecture that exhibits an adaptive response to pH. The system was fabricated by microfluidic generation of semipermeable biomimetic polymer vesicles within 1 min using oleyl alcohol as the oil phase. The polymersomes allowed for the diffusion of protons and substrates acting as external signals. Using this method, we were able to construct adaptive microreactors containing internal polyelectrolyte-based catalytic organelles capable of sequestering and localizing enzymes and reaction products in a dynamic process driven by an external stimulus. This approach provides a platform for the rapid and efficient construction of robust adaptive microreactors that can be used in catalysis, biosensing, and cell mimicry.

Bursting and Reassembly of Giant Double Emulsion Drops Form Polymer Vesicles.

Polymeric vesicles are excellent building blocks of synthetic compartmentalized systems such as protocells and artificial organelles. In such applications, the efficient encapsulation of materials into the vesicles is an essential requirement. However, common encapsulation techniques can be time-consuming, demand special equipment or have limited efficiency for large components, such as proteins and nanoparticles. Here, we describe a simple method to create cargo-filled polymer vesicles based on bursting and reassembly of giant double emulsion droplets (DED). Due to their large average diameter of 2 mm, DEDs eventually burst in the aqueous medium, producing polymeric film fragments. These fragments rapidly reassemble into smaller vesicles in a process involving folding, fusion and vesiculation. The daughter vesicles have an average diameter of 10 µm, representing a two-order of magnitude size reduction compared to the original DED, and can efficiently encapsulate components present in solution by entrapment of the aqueous medium during vesicle reassembly.

Artificial Organelles for Energy Regeneration.

One of the critical steps in sustaining life-mimicking processes in synthetic cells is energy, i.e., adenosine triphosphate (ATP) regeneration. Previous studies have shown that the simple addition of ATP or ATP regeneration systems, which do not regenerate ATP directly from ADP and Pi , have no or only limited success due to accumulation of ATP hydrolysis products. In general, ATP regeneration can be achieved by converting light or chemical energy into ATP, which may also involve redox transformations of cofactors. The present contribution provides an overview of the existing ATP regeneration strategies and the related nicotinamide adenine dinucleotide (NAD+ ) redox cycling, with a focus on compartmentalized systems. Special attention is being paid to those approaches where so-called artificial organelles are developed. They comprise a semipermeable membrane functionalized by biological or man-made components and employ external energy in the form of light or nutrients in order to generate a transmembrane proton gradient, which is further utilized for ATP synthesis.