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Hybrid Protocells

Hybrid Protocells

Although coacervate micro-droplets can be stabilized by excess surface charge, under many conditions they exhibit a propensity to coalesce into larger droplets or undergo macroscopic phase separation. As a consequence, their use as a protocell model can be compromised by their liquid-like behaviour and low surface tension. To circumvent this problem, and to generate a new type of hybrid protocell, we have recently developed several methods to produce membrane-bounded coacervate micro-droplets via spontaneous assembly or partitioning of auxiliary components on the surface of the liquid micro-compartments.

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Key references:

Fatty Acid Membrane Assembly on Coacervate Micro-droplets as a Step towards a Hybrid Protocell Model:

We are exploring the use of simple physical and chemical processes to generate new types of hybrid protocells based on the spontaneous self-assembly of a continuous fatty acid multilamellar membrane at the surface of preformed coacervate microdroplets that comprise molecularly crowded interiors. The approach integrates notions of membrane-mediated compartmentalization, chemical enrichment and internalized structuration, and brings together alternative models of prebiotic compartmentalization based on either phase separated liquid droplets or fatty acid vesicle self-assembly. Our studies indicate that the membrane-enclosed coacervate micro-compartments are semipermeable and exhibit uptake and exclusion properties different to those of the uncoated microdroplets. Thus, it should be possible to exploit the hybrid protocell model to develop transmembrane control of coacervate-sequestered reaction systems such as gene-directed protein synthesis or RNA catalysis by diffusion-limited regulation of the uptake of small molecules from the external continuous aqueous phase.

Small-molecule Uptake in Membrane-free Peptide/ nucleotide Protocells:

Our investigations show that the anionic fluorescent dye 8-anilionapthalene sulphonate (ANS) can be readily sequestered into the interior of positively charged oligolysine/ATP coacervate micro-droplets to produce a dispersed microphase that exhibits increased morphological stability and complex internal structuration. The uptake of ANS into oligolysine/ATP coacervate microdroplets occurs initially in the outer regions of the droplets, and is associated with a progressive reduction in the zeta potential of the dispersion due to formation of loosely associated ANS/oligolysine/ATP complexes that retain the polar environment and dynamical fluctuations of the native peptide/nucleotide matrix.

Key references:

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Key references:

Spontaneous Structuration in Coacervate-based Protocells by Polyoxometalate-mediated Membrane Assembly:

We are also interested in developing hybrid protocells that consist of coacervate micro-droplets enclosed within an inorganic-based membrane. Recently, we demonstrated that molecularly crowded polyelectrolyte/ATP-enriched coacervate droplets can be transformed into membrane-bounded vesicles by using a polyoxometalate-mediated surface-templating procedure. The coacervate to vesicle transition results in spontaneous structuration of the coacervate micro-droplets into novel three-tiered micro-compartments comprising a semi-permeable negatively charged polyoxometalate/polyelectrolyte outer membrane typically 600 nm in thickness, a 2–5 μm-wide sub-membrane coacervate shell capable of sequestering organic dyes, proteins and nanoparticles, and an internal aqueous lumen often tens of micrometres in diameter. The encapsulated proteins are inaccessible to proteases in the external medium, and can be exploited for the spatial localization and coupling of enzyme cascade reactions that are generated within single or between multiple populations of the coacervate vesicles. In particular, by locating different enzymes in different protocell populations, we produce enclosed reaction compartments that can communicate chemically at a distance, provided that the reaction intermediates can diffuse across the boundary membrane.

Spatial positioning and chemical coupling in coacervate-in-proteinosome protocells:

The integration of molecularly crowded micro-environments into membrane-enclosed protocell models represents a step towards more realistic representations of cellular structure and organization. Herein, the membrane diffusion-mediated nucleation of negatively or positively charged coacervate micro-droplets within the aqueous lumen of individual proteinosomes is used to prepare nested hybrid protocells with spatially organized and chemically coupled enzyme activities. The location and reconfiguration of the entrapped droplets are regulated by tuning the electrostatic interactions between the encapsulated coacervate and surrounding negatively charged proteinosome membrane. As a consequence, alternative modes of a cascade reaction involving membrane- and coacervate-segregated enzymes can be implemented within the coacervate-in-proteinosome protocells.    

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Key references:

  • Richard Booth, Yan Qiao, Mei Li, Stephen Mann, Angew. Chem. Int. Ed., 2019, DOI: 10.1002/anie.201903756


Key references:

  • Liu S, Zhang Y, Li M, Xiong L, Yang X, He X, Wang K, Liu J and Mann S, Nature Chemistry, 12, 1165-1173 (2020)

Enzyme-mediated nitric oxide production in vasoactive erythrocyte membrane-enclosed coacervate protocells:

The design and construction of synthetic therapeutic protocells capable of establishing cognate chemical communication channels with living cells is an important challenge for synthetic biology and bio-engineering. Here we develop a step towards protocell-mediated nitric-oxide-induced vasodilation by constructing a new synthetic cell model based on bio-derived coacervate vesicles with high haemocompatibility and increased blood circulation times. The hybrid protocells are prepared by the spontaneous self-assembly of haemoglobin-containing erythrocyte membrane fragments on the surface of preformed DEAE-dextran/dsDNA coacervate micro-droplets containing glucose oxidase. We use the sequestered enzymes to program a spatially coupled glucose oxidase/haemoglobin reaction cascade, which in the presence of glucose and hydroxyurea generates a protocell-mediated flux of nitric oxide that we exploit for in vitro and in vivo blood vessel vasodilation. Taken together, our results provide new opportunities for the development of endogenously organized cell-like entities (biocompatible micro-bots) geared specifically towards active interfacing with individual living cells and cell communities.