Catalytic processing in ruthenium-based polyoxometalate coacervate protocells

ARTICLE https://doi.org/10.1038/s41467-019-13759-1 OPEN Catalytic processing in ruthenium-based polyoxometalate coacervate protocells 1234567890():,; Pierangelo Gobbo 1, Liangfei Tian Avinash J. Patil 1*, Mauro Carraro 1,4, B.V.V.S Pavan Kumar 3, Marcella Bonchio 1,4, Samuel Turvey1, Mattia Cattelan 2, 3* & Stephen Mann1* The development of programmable microscale materials with cell-like functions, dynamics and collective behaviour is an important milestone in systems chemistry, soft matter bioengineering and synthetic protobiology. Here, polymer/nucleotide coacervate microdroplets are reconfigured into membrane-bounded polyoxometalate coacervate vesicles (PCVs) in the presence of a bio-inspired Ru-based polyoxometalate catalyst to produce synzyme protocells (Ru4PCVs) with catalase-like activity. We exploit the synthetic protocells for the implementation of multi-compartmentalized cell-like models capable of collective synzyme-mediated buoyancy, parallel catalytic processing in individual horseradish peroxidase-containing Ru4PCVs, and chemical signalling in distributed or encapsulated multicatalytic protocell communities. Our results highlight a new type of catalytic microcompartment with multi-functional activity and provide a step towards the development of protocell reaction networks. 1 Centre for Organized Matter Chemistry and Centre for Protolife Research, School of Chemistry, University of Bristol, Bristol BS8 1TS, UK. 2 School of Chemistry, University of Bristol, Bristol BS8 1TS, UK. 3 ITM-CNR and Dipartimento di Scienze Chimiche, Università di Padova, Via F. Marzolo 1, 35131 Padova, Italy. 4These authors contributed equally: Liangfei Tian, B. V. V. S Pavan Kumar. *email: ; ; NATURE COMMUNICATIONS | (2020)11:41 | https://doi.org/10.1038/s41467-019-13759-1 | www.nature.com/naturecommunications 1 ARTICLE T NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13759-1 he endogenous operation and integration of chemical processes within aqueous filled micro-compartments is providing opportunities for the development of programmable microscale materials with cell-like functions, dynamics and collective behaviors. Membrane-bounded synthetic protocells can be produced in the form of lipid vesicles1–3, polymersomes4–7, polypeptide capsules8,9, dendrimersomes10,11, inorganic colloidosomes12–14, semi-permeable protein–polymer microcapsules (proteinosomes)15–18, and semi-permeable bio-inorganic microcapsules19. In addition, coacervate micro-droplets are being exploited as membrane-free protocell models as they selectively sequester key functional components such as biomolecular substrates, proteins, polynucleotides, ribozymes, ribosomes, and chloroplasts20–23, and support photochemical reactions, enzyme/ ribozyme cascades and gene expression within their molecularly crowded interior24–28. As protocell models, coacervate droplets can be stabilized by enclosure within fatty acid or block copolymer membranes29,30, endocytosed within liposomes31 and proteinosomes32,33, enlisted as predatory protocells34, exploited as functional modules in prototissues35 and sensing platforms36, and reconfigured into fatty acid vesicles32,37 or membranebounded coacervate vesicles38. In the latter case, coacervate droplets prepared from polydiallydimethylammonium chloride (PDDA) and adenosine 5′-triphosphate (ATP) were transformed by electrostatically induced complexation of a polyoxometalate (POM) (sodium phosphotungstate, [PW11O39]7−; PTA) with PDDA molecules present at the droplet surface. The concomitant changes in osmotic pressure gave rise to a complex three-tiered microstructure comprising a semi-permeable negatively charged PTA/PDDA outer membrane, a sub-membrane coacervate shell containing guest components, and an internal aqueous lumen38. Although the resulting POM coacervate vesicles (PCVs) exhibited no intrinsic chemical reactivity, they could be rendered functional by encapsulation of enzymes within the coacervate submembrane layer. In this paper, we extend the above approach to the spontaneous preparation of catalytic PCVs by transforming the PDDA/ ATP coacervate droplets in the presence of mixture of PTA and a bio-inspired Ru(IV)-based POM polyanionic catalyst (Na10[Ru4(μ–O)4(μ–OH)2(H2O)4(γ–SiW10O36)2]; Ru4POM)39,40 to produce synzyme active protocells (Ru4PCVs) with catalaselike membrane activity. We use Ru4POM as an exogenous agent as it is readily synthesized, chemically stable, and forms a strong electrostatic complex with PDDA polycations present at the droplet surface due to its high negative charge. Moreover, the adamantane-like Ru-oxo core of the synzyme comprises four redox active sites connected through μ-oxo and μ-hydroxo bridges, which mimic the natural oxygen evolving photosynthetic center to produce an effective bio-inspired oxygenic catalyst41,42. We assess the synzyme activity of the membrane-integrated Ru4POM structural unit by determining the levels of O2 produced when populations of the Ru4PCVs are incubated with aqueous H2O2, and exploit the collective catalase-like activity to design a multi-compartmentalized protocell model capable of endogenously driven buoyancy. By incorporating competitive synzyme and peroxidase reaction pathways within individual protocells we prepare spatially organized networks of Ru4PCVs that undergo parallel catalytic processing. Alternatively, we use the same competitive reactions to implement a spatially distributed signaling pathway within a ternary protocell community dispersed in aqueous medium or encapsulated within water-in-oil emulsion droplets. In both cases, the consortium consists of a transmitter population of synzyme-inactive glucose oxidase (GOx)-containing PCVs that endogenously produce a H2O2 signal and two competitive receiver populations of peroxidase-active or synzyme-active PCVs and Ru4PCVs, respectively. Taken together, 2 our results provide opportunities for the fabrication of new types of catalytic micro-compartments based on membrane-integrated POM clusters and provide a step towards the development of protocell reaction networks. Results Catalytic activity of synzyme protocells. Membrane-free PDDA/ ATP coacervate micro-droplets were structurally and compositionally reconfigured into membrane-bounded coacervate vesicles by addition of an aqueous solution of Ru4POM and PTA polyanions (Fig. 1a). Typically, Ru4PCVs prepared at a PTA: Ru4POM molar ratio of 7:1 were intact, non-aggregated, birefringent, and polydisperse (mean diameter, 25 ± 15 µm; 30 s stirring time) (Fig. 1b–d and Supplementary Fig. 1). SEM images confirmed a hollow interior and smooth pliant outer membrane, 500–800 nm in thickness (Fig. 1e and Supplementary Fig. 2), which was consistent with a three-tiered microstructure as described previously for PTA-CVs38. Ru4PCVs with larger sizes and increased polydispersity were obtained by increasing the extent of coacervate droplet coalescence prior to reconfiguration (Supplementary Fig (...truncated)