Complex Coacervate Materials as Artificial Cells

Cells have evolved to be self-sustaining compartmentalized systems that consist of many thousands of biomolecules and metabolites interacting in complex cycles and reaction networks. Numerous subtle intricacies of these self-assembled structures are still largely unknown. The importance of liquid-liquid phase separation (both membraneless and membrane bound) is, however, recognized as playing an important role in achieving biological function that is controlled in time and space. Reconstituting biochemical reactions in vitro has been a success of the last decades, for example, establishment of the minimal set of enzymes and nutrients able to replicate cellular activities like the in vitro transcription translation of genes to proteins. Further than this though, artificial cell research has the aim of combining synthetic materials and nonliving macromolecules into ordered assemblies with the ability to carry out more complex and ambitious cell-like functions. These activities can provide insights into fundamental cell processes in simplified and idealized systems but could also have an applied impact in synthetic biology and biotechnology in the future. To date, strategies for the bottom-up fabrication of micrometer scale life-like artificial cells have included stabilized water-in-oil droplets, giant unilamellar vesicles (GUV’s), hydrogels, and complex coacervates. Water-in-oil droplets are a valuable and easy to produce model system for studying cell-like processes; however, the lack of a crowded interior can limit these artificial cells in mimicking life more closely. Similarly membrane stabilized vesicles, such as GUV’s, have the additional membrane feature of cells but still lack a macromolecularly crowded cytoplasm. Hydrogel-based artificial cells have a macromolecularly dense interior (although cross-linked) that better mimics cells, in addition to mechanical properties more similar to the viscoelasticity seen in cells but could be seen as being not dynamic in nature and limiting to the diffusion of biomolecules. On the other hand, liquid-liquid phase separated complex coacervates are an ideal platform for artificial cells as they can most accurately mimic the crowded, viscous, highly charged nature of the eukaryotic cytoplasm. Other important key features that researchers in the field target include stabilizing semipermeable membranes, compartmentalization, information transfer/communication, motility, and metabolism/growth. In this Account, we will briefly cover aspects of coacervation theory and then outline key cases of synthetic coacervate materials used as artificial cells (ranging from polypeptides, modified polysaccharides, polyacrylates, and polymethacrylates, and allyl polymers), finishing with envisioned opportunities and potential applications for coacervate artificial cells moving forward.</p

Functional interactions between bottom-up synthetic cells and living matter for biomedical applications

Bottom-up synthetic cells, where diverse non-living materials are combined in creative ways in order to construct increasingly life-like and adaptive systems, are fast approaching a level of function that will enable significant advances in solving specific biomedical challenges. Over the last 10 years, we have seen a wide variety of synthetic cell based approaches to challenges in regulating antimicrobial activity, delivering cargo to mammalian cells, and “growth support”. Despite this progress, there has not been a widespread uptake of synthetic cell technologies in biomedical engineering. In this Review, we highlight both the strengths and limitations of these existing synthetic cell applications, as well as give an overview of the state-of-the-art of synthetic cell technology that has yet been applied to cellular contexts. In doing so we aim to identify opportunities for the advancement of this unique intersection of research fields

Tuning of Cationic Polymer Functionality in Complex Coacervate Artificial Cells for Optimized Enzyme Activity

Complex coacervates are a versatile platform to mimic the structure of living cells. In both living systems and artificial cells, a macromolecularly crowded condensate phase has been shown to be able to modulate enzyme activity. Yet, how enzyme activity is affected by interactions (particularly with cationic charges) inside coacervates is not well studied. Here, we synthesized a series of amino-functional polymers to investigate the effect of the type of amine and charge density on coacervate formation, stability, protein partitioning, and enzyme function. The polymers were prepared by RAFT polymerization using as monomers aminoethyl methacrylate (AEAM), 2-(dimethylamino)ethyl methacrylate (DMAEMA), imidazolepropyl methacrylamide (IPMAm), and [2-(methacryloyloxy)ethyl] trimethylammonium chloride (TMAEMA). Membranized complex coacervate artificial cells were formed with these polycations and an anionic amylose derivative. Results show that polycations with reduced charge density result in higher protein mobility in the condensates and also higher enzyme activity. Insights described here could help guide the use of coacervate artificial cells in applications such as sensing, catalysis, and therapeutic formulations.</p

Amphiphilic AIEgen-polymer aggregates: Design, self-assembly and biomedical applications

Aggregation-induced emission (AIE) is a phenomenon in which fluorescence is enhanced rather than quenched upon molecular assembly. AIE fluorogens (AIEgens) are flexible, conjugated systems that are limited in their dynamics when assembled, which improves their fluorescent properties. This intriguing feature has been incorporated in many different molecular assemblies and has been extended to nanoparticles composed of amphiphilic polymer building blocks. The integration of the fascinating AIE design principle with versatile polymer chemistry opens up new frontiers to approach and solve intrinsic obstacles of conventional fluorescent materials in nanoscience, including the aggregation-caused quenching effect. Furthermore, this integration has drawn significant attention from the nanomedicine community, due to the additional advantages of nanoparticles comprising AIEgenic molecules, such as emission brightness and fluorescence stability. In this regard, a range of AIEgenic amphiphilic polymers have been developed, displaying enhanced emission in the self-assembly/aggregated state. AIEgenic assemblies are regarded as attractive nanomaterials with inherent fluorescence, which display promising features in a biomedical context, for instance in biosensing, cell/tissue imaging and tracking, as well as (photo) therapeutics. In this review, we describe recent strategies for the design and synthesis of novel types of AIEgenic amphiphilic polymers via facile approaches including direct conjugation to natural/synthetic polymers, polymerization, post-polymerization and supramolecular host−guest interactions. Their self-assembly behavior and biomedical potential will be discussed

Dampened Transient Actuation of Hydrogels Autonomously Controlled by pH-Responsive Bicontinuous Nanospheres

The fabrication of a soft actuator with a dampened actuation response is presented. This was achieved via the incorporation into an actuating hydrogel of urease-loaded pH-responsive bicontinuous nanospheres (BCNs), whose membrane was able to regulate the permeability and thus conversion of fuel urea into ammonia. The dampened response of these nanoreactors to the enzymatically induced pH change was translated to a pH-responsive soft actuator. In hydrogels composed of a pH-responsive and nonresponsive layer, the transient pH gradient yielded an asymmetric swelling behavior, which induced a bending response. The transient actuation profile could be controlled by varying the external fuel concentrations. Furthermore, we showed that the spatial organization of the BCNs within the actuator had a great influence on the actuation response. Embedding the urease-loaded nanoreactors within the active, pH-responsive layer resulted in a reduced response due to local substrate conversion in comparison to embedding them within the passive layer of the bilayer hydrogel. Finally, we were able to induce transient actuation in a hydrogel comprising two identical active layers by the immobilization of the BCNs within one specific layer. Upon addition of urea, a local pH gradient was generated, which caused accelerated swelling in the BCN layer and transient bending of the device before the pH gradient was attenuated over time.</p

Chemical Cascading Between Polymersomal Nanoreactor Populations

[EN] Harnessing interactions of functional nano-compartments to generate larger particle assemblies allows studying diverse biological behaviors based on their population states and can lead to the development of smart materials. Herein, thiol-functionalized polymersome nanoreactors are utilized as responsive organelle-like nano-compartments-with inherent capacity to associate into larger aggregates in response to change in the redox state of their environment-to study the kinetics of cascade reactions and explore functions of their collective under different population states. Two nanoreactor populations, glucose oxidase- and horseradish peroxidase-loaded polymersomes, are prepared, and the results of their cascading upon addition of glucose are investigated. The kinetics of resorufin production in associated polymersomes and non-associated polymersome populations are compared, observing a decreased rate upon association. For the associated populations, faster chemical cascading is found when the two types of nanoreactors are associated in a concerted step, as compared to sequential association. The addition of competing agents such as catalase impacts the communication between non-associated polymersomes, whereas such an effect is less pronounced for the associated ones. Altogether, the results showcase the impact of collective associations on enzymatic cascading between organelle-like nanoreactors.Y.A. and A.L.-L. contributed equally to this work. The authors would like to acknowledge the support from the Dutch Ministry of Education, Culture, and Science (Gravitation program 024.001.035 and Spinoza premium) and the ERC Advanced Grant (Artisym 694120).A.L.-L. acknowledges support from the MSCA Cofund project oLife, which has received funding from the European Union's Horizon 2020 research and innovation program under the Grant Agreement 847675; and the Maria Zambrano Program from the Spanish Government funded by NextGenerationEU from the European Union. Dr. Imke Pijpers is thanked for cryo-TEM imaging. Dr. Pascal Welzen is acknowledged for advice and useful discussion on polymer and polymersome preparation.Altay, Y.; Llopis-Lorente, A.; Abdelmohsen, LKEA.; Van Hest, JC. (2023). Chemical Cascading Between Polymersomal Nanoreactor Populations. Macromolecular Chemistry and Physics. 224(1):1-5. https://doi.org/10.1002/macp.20220026915224

Models of Chemical Communication for Micro/Nanoparticles

Engineering chemical communication between micro/nanosystems (via the exchange of chemical messengers) is receiving increasing attention from the scientific community. Although a number of micro- and nanodevices (e.g., drug carriers, sensors, and artificial cells) have been developed in the last decades, engineering communication at the micro/nanoscale is a recent emergent topic. In fact, most of the studies in this research area have been published within the last 10 years. Inspired by nature─where information is exchanged by means of molecules─the development of chemical communication strategies holds wide implications as it may provide breakthroughs in many areas including nanotechnology, artificial cell research, biomedicine, biotechnology, and ICT. Published examples rely on nanotechnology and synthetic biology for the creation of micro- and nanodevices that can communicate. Communication enables the construction of new complex systems capable of performing advanced coordinated tasks that go beyond those carried out by individual entities. In addition, the possibility to communicate between synthetic and living systems can further advance our understanding of biochemical processes and provide completely new tailored therapeutic and diagnostic strategies, ways to tune cellular behavior, and new biotechnological tools. In this Account, we summarize advances by our laboratories (and others) in the engineering of chemical communication of micro- and nanoparticles. This Account is structured to provide researchers from different fields with general strategies and common ground for the rational design of future communication networks at the micro/nanoscale. First, we cover the basis of and describe enabling technologies to engineer particles with communication capabilities. Next, we rationalize general models of chemical communication. These models vary from simple linear communication (transmission of information between two points) to more complex pathways such as interactive communication and multicomponent communication (involving several entities). Using illustrative experimental designs, we demonstrate the realization of these models which involve communication not only between engineered micro/nanoparticles but also between particles and living systems. Finally, we discuss the current state of the topic and the future challenges to be addressed.</p

Inherently Fluorescent Peanut-Shaped Polymersomes for Active Cargo Transportation

Nanomotors have been extensively explored for various applications in nanomedicine, especially in cargo transportation. Motile properties enable them to deliver pharmaceutical ingredients more efficiently to the targeted site. However, it still remains a challenge to design motor systems that are therapeutically active and can also be effectively traced when taken up by cells. Here, we designed a nanomotor with integrated fluorescence and therapeutic potential based on biodegradable polymersomes equipped with aggregation-induced emission (AIE) agents. The AIE segments provided the polymersomes with autofluorescence, facilitating the visualization of cell uptake. Furthermore, the membrane structure enabled the reshaping of the AIE polymersomes into asymmetric, peanut-shaped polymersomes. Upon laser irradiation, these peanut polymersomes not only displayed fluorescence, but also produced reactive oxygen species (ROS). Because of their specific shape, the ROS gradient induced motility in these particles. As ROS is also used for cancer cell treatment, the peanut polymersomes not only acted as delivery vehicles but also as therapeutic agents. As an integrated platform, these peanut polymersomes therefore represent an interesting delivery system with biomedical potential.</p

Compartmentalized Intracellular Click Chemistry with Biodegradable Polymersomes

Polymersome nanoreactors that can be employed as artificial organelles have gained much interest over the past decades. Such systems often include biological catalysts (i.e., enzymes) so that they can undertake chemical reactions in cellulo. Examples of nanoreactor artificial organelles that acquire metal catalysts in their structure are limited, and their application in living cells remains fairly restricted. In part, this shortfall is due to difficulties associated with constructing systems that maintain their stability in vitro, let alone the toxicity they impose on cells. This study demonstrates a biodegradable and biocompatible polymersome nanoreactor platform, which can be applied as an artificial organelle in living cells. The ability of the artificial organelles to covalently and non-covalently incorporate tris(triazolylmethyl)amine-Cu(I) complexes in their membrane is shown. Such artificial organelles are capable of effectively catalyzing a copper-catalyzed azide-alkyne cycloaddition intracellularly, without compromising the cells’ integrity. The platform represents a step forward in the application of polymersome-based nanoreactors as artificial organelles

Regulating Chemokine-Receptor Interactions through the Site-Specific Bioorthogonal Conjugation of Photoresponsive DNA Strands

Oligonucleotide conjugation has emerged as a versatile molecular tool for regulating protein activity. A state-of-the-art labeling strategy includes the site-specific conjugation of DNA, by employing bioorthogonal groups genetically incorporated in proteins through unnatural amino acids (UAAs). The incorporation of UAAs in chemokines has to date, however, remained underexplored, probably due to their sometimes poor stability following recombinant expression. In this work, we designed a fluorescent stromal-derived factor-1β (SDF-1β) chemokine fusion protein with a bioorthogonal functionality amenable for click reactions. Using amber stop codon suppression, p-azido-L-phenylalanine was site-specifically incorporated in the fluorescent N-terminal fusion partner, superfolder green fluorescent protein (sfGFP). Conjugation to single-stranded DNAs (ssDNA), modified with a photocleavable spacer and a reactive bicyclononyne moiety, was performed to create a DNA-caged species that blocked the receptor binding ability. This inhibition was completely reversible by means of photocleavage of the ssDNA strands. The results described herein provide a versatile new direction for spatiotemporally regulating chemokine-receptor interactions, which is promising for tissue engineering purposes.</p