Stephen Mann

Fatty acid membrane assembly on coacervate microdroplets as a step towards a hybrid protocell model

Mechanisms of prebiotic compartmentalization are central to providing insights into how protocellular systems emerged on the early Earth. Protocell models are based predominantly on the membrane self-assembly of fatty-acid vesicles, although membrane-free scenarios that involve liquid-liquid microphase separation (coacervation) have also been considered. Here we integrate these alternative models of prebiotic compartmentalization and develop a hybrid protocell model based on the spontaneous self-assembly of a continuous fatty-acid membrane at the surface of preformed coacervate microdroplets prepared from cationic peptides/polyelectrolytes and adenosine triphosphate or oligo/polyribonucleotides. We show that the coacervate-supported membrane is multilamellar, and mediates the selective uptake or exclusion of small and large molecules. The coacervate interior can be disassembled without loss of membrane integrity, and fusion and growth of the hybrid protocells can be induced under conditions of high ionic strength. Our results highlight how notions of membrane-mediated compartmentalization, chemical enrichment and internalized structuration can be integrated in protocell models via simple chemical and physical processes.

Synthetic cellularity based on non-lipid micro-compartments and protocell models

This review discusses recent advances in the design and construction of protocell models based on the self-assembly or microphase separation of non-lipid building blocks. We focus on strategies involving partially hydrophobic inorganic nanoparticles (colloidosomes), protein-polymer globular nano-conjugates (proteinosomes), amphiphilic block copolymers (polymersomes), and stoichiometric mixtures of oppositely charged biomolecules and polyelectrolytes (coacervates). Developments in the engineering of membrane functionality to produce synthetic protocells with gated responses and control over multi-step reactions are described. New routes to protocells comprising molecularly crowded, cytoskeletal-like hydrogel interiors, as well as to the construction of hybrid protocell models are also highlighted. Together, these strategies enable a wide range of biomolecular and synthetic components to be encapsulated, regulated and processed within the micro-compartmentalized volume, and suggest that the development of non-lipid micro-ensembles offers an approach that is complementary to protocell models based on phospholipid or fatty acid vesicles.

Directing chondrogenesis of stem cells with specific blends of cellulose and silk

Biomaterials that can stimulate stem cell differentiation without growth factor supplementation provide potent and cost-effective scaffolds for regenerative medicine. We hypothesize that a scaffold prepared from cellulose and silk blends can direct stem cell chondrogenic fate. We systematically prepared cellulose blends with silk at different compositions using an environmentally benign processing method based on ionic liquids as a common solvent. We tested the effect of blend compositions on the physical properties of the materials as well as on their ability to support mesenchymal stem cell (MSC) growth and chondrogenic differentiation. The stiffness and tensile strength of cellulose was significantly reduced by blending with silk. The characterized materials were tested using MSCs derived from four different patients. Growing MSCs on a specific blend combination of cellulose and silk in a 75:25 ratio significantly upregulated the chondrogenic marker genes SOX9, aggrecan, and type II collagen in the absence of specific growth factors. This chondrogenic effect was neither found with neat cellulose nor the cellulose/silk 50:50 blend composition. No adipogenic or osteogenic differentiation was detected on the blends, suggesting that the cellulose/silk 75:25 blend induced specific stem cell differentiation into the chondrogenic lineage without addition of the soluble growth factor TGF-β. The cellulose/silk blend we identified can be used both for in vitro tissue engineering and as an implantable device for stimulating endogenous stem cells to initiate cartilage repair.

Microfluidic Formation of Membrane-Free Aqueous Coacervate Droplets in Water

We report on the formation of coacervate droplets from poly(diallyldimethylammonium chloride) with either adenosine triphosphate or carboxymethyl-dextran using a microfluidic flow-focusing system. The formed droplets exhibit improved stability and narrower size distributions for both coacervate compositions when compared to the conventional vortex dispersion techniques. We also demonstrate the use of two parallel flow-focusing channels for the simultaneous formation and co-location of two distinct populations of coacervate droplets containing different DNA oligonucleotides, and that the populations can coexist in close proximity up to 48 h without detectable exchange of genetic information. Our results show that the observed improvements in droplet stability and size distribution may be scaled with ease. In addition, the ability to encapsulate different materials into coacervate droplets using a microfluidic channel structure allows for their use as cell-mimicking compartments.

Non-equilibrium behaviour in coacervate-based protocells under electric-field-induced excitation.

Although numerous strategies are now available to generate rudimentary forms of synthetic cell-like entities, minimal progress has been made in the sustained excitation of artificial protocells under non-equilibrium conditions. Here we demonstrate that the electric field energization of coacervate microdroplets comprising polylysine and short single strands of DNA generates membrane-free protocells with complex, dynamical behaviours. By confining the droplets within a microfluidic channel and applying a range of electric field strengths, we produce protocells that exhibit repetitive cycles of vacuolarization, dynamical fluctuations in size and shape, chaotic growth and fusion, spontaneous ejection and sequestration of matter, directional capture of solute molecules, and pulsed enhancement of enzyme cascade reactions. Our results highlight new opportunities for the study of non-equilibrium phenomena in synthetic protocells, provide a strategy for inducing complex behaviour in electrostatically assembled soft matter microsystems and illustrate how dynamical properties can be activated and sustained in microcompartmentalized media.

Enzyme activity in liquid lipase melts as a step towards solvent-free biology at 150 °C

Water molecules play a number of critical roles in enzyme catalysis, including mass transfer of substrates and products, nucleophilicity and proton transfer at the active site, and solvent shell-mediated dynamics for accessing catalytically competent conformations. The pervasiveness of water in enzymolysis therefore raises the question concerning whether biocatalysis can be undertaken in the absence of a protein hydration shell. Lipase-mediated catalysis has been undertaken with reagent-based solvents and lyophilized powders, but there are no examples of molecularly dispersed enzymes that catalyse reactions at sub-solvation levels within solvent-free melts. Here we describe the synthesis, properties and enzyme activity of self-contained reactive biofluids based on solvent-free melts of lipase-polymer surfactant nanoconjugates. Desiccated substrates in liquid (p-nitrophenyl butyrate) or solid (p-nitrophenyl palmitate) form can be mixed or solubilized, respectively, into the enzyme biofluids, and hydrolysed in the solvent-free state. Significantly, the efficiency of product formation increases as the temperature is raised to 150 °C.

Small-molecule uptake in membrane-free peptide/nucleotide protocells

The spontaneous phase separation of peptide/nucleotide droplets in water produces membrane-free chemically organized micro-compartments that offer new opportunities for the construction of synthetic cells and development of protocell models of prebiotic organization. Certain small molecules can be sequestered into the droplet interior but the uptake mechanisms are unexplored. Using confocal fluorescence microscopy, 31P NMR spectroscopy, fluorescence spectroscopy and lateral molecular force microscopy, we probe the molecular interactions associated with sequestration of the water-soluble fluorescent anionic dye 1-anilinonapthalene-8-sulphonic acid (ANS) into positively charged oligolysine/ATP coacervate micro-droplets. Our results indicate that uptake of ANS proceeds initially through electrostatic interactions involving a ternary ANS/oligolysine/ATP complex, followed by a secondary mechanism based on non-polar interactions between ANS and ATP. We demonstrate that at very high levels of ANS the hybrid droplets develop a thin outer shell that is mechanically more compliant than the droplet interior, and acts as a quasi-membrane for restricting the influx of methylene blue. Our results suggest that understanding the mechanisms of molecular uptake into coacervate droplets could provide an important step towards the rational design of molecularly crowded microscale dispersions that display complex fluid behavior, compartment-mediated functionality and primitive aspects of synthetic cellularity.

Hydrophobic nanoparticles promote lamellar to inverted hexagonal transition in phospholipid mesophases.

This study focuses on how the mesophase transition behaviour of the phospholipid dioleoyl phosphatidylethanolamine (DOPE) is altered by the presence of 10 nm hydrophobic and 14 nm hydrophilic silica nanoparticles (NPs) at different concentrations. The lamellar to inverted hexagonal phase transition (Lα-HII) of phospholipids is energetically analogous to the membrane fusion process, therefore understanding the Lα-HII transition with nanoparticulate additives is relevant to how membrane fusion may be affected by these additives, in this case the silica NPs. The overriding observation is that the HII/Lα boundaries in the DOPE p-T phase diagram were shifted by the presence of NPs: the hydrophobic NPs enlarged the HII phase region and thus encouraged the inverted hexagonal (HII) phase to occur at lower temperatures, whilst hydrophilic NPs appeared to stabilise the Lα phase region. This effect was also NP-concentration dependent, with a more pronounced effect for higher concentration of the hydrophobic NPs, but the trend was less clear cut for the hydrophilic NPs. There was no evidence that the NPs were intercalated into the mesophases, and as such it was likely that they might have undergone microphase separation and resided at the mesophase domain boundaries. Whilst the loci and exact roles of the NPs invite further investigation, we tentatively discuss these results in terms of both the surface chemistry of the NPs and the effect of their curvature on the elastic bending energy considerations during the mesophase transition.

Higher-order assembly of crystalline cylindrical micelles into membrane-extendable colloidosomes

Crystallization-driven self-assembly of diblock copolymers into cylindrical micelles of controlled length has emerged as a promising approach to the fabrication of functional nanoscale objects with high shape anisotropy. Here we show the preparation of a series of crystallizable diblock copolymers with appropriate wettability and chemical reactivity, and demonstrate their self-assembly into size-specific cylindrical micelle building blocks for the hierarchical construction of mechanically robust colloidosomes with a range of membrane textures, surface chemistries and optical properties. The colloidosomes can be structurally elaborated post assembly by in situ epitaxial elongation of the membrane building blocks to produce microcapsules covered in a chemically distinct, dense network of hair-like outgrowths. Our approach provides a route to hierarchically ordered colloidosomes that retain the intrinsic growth activity of their constituent building blocks to permit biofunctionalization, and have potential applications in areas such as biomimetic encapsulation, drug delivery, catalysis and biosensing.Functional nanoscale objects can be prepared via crystallization-driven self-assembly of diblock copolymers. Here the authors show the self-assembly of crystalline block copolymers into size-specific cylindrical micelles for the hierarchical construction of mechanically robust colloidosomes with a range of membrane textures.

Dynamic Behavior in Enzyme–Polymer Surfactant Hydrogel Films

Dynamic protein-polymer surfactant films are highly hydrophilic and show a soft solid to hydrogel transition upon hydration to produce a swollen hydrogel. An unusual reversible autospreading/self-folding response is observed when the water-saturated films are transferred from water into air.