Spontaneous Membranization in a Silk-Based Coacervate Protocell Model.

Molecularly crowded coacervate micro-droplets are useful protocell constructs but the absence of a physical membrane limits their application as cytomimetic models. Auxiliary surface-active agents have been harnessed to stabilize the coacervate droplets by irreversible shell formation but endogenous processes of reversible membranization have received minimal attention. Herein, we describe a dynamic alginate/silk coacervate-based protocell model in which membrane-less droplets are reversibly reconfigured and inflated into semipermeable coacervate vesicles by spontaneous self-organization of amphiphilic silk polymers at the droplet surface under non-neutral charge conditions in the absence of auxiliary agents. We show that membranization can be reversibly controlled endogenously by programming the pH within the protocells using an antagonistic enzyme system such that structural reconfigurations in the protocell microstructure are coupled to the trafficking of water-soluble solutes. Our results open new perspectives in the design of hybrid protocell models with dynamical structural properties.

Programmed assembly of synthetic protocells into thermoresponsive prototissues.

Although several new types of synthetic cell-like entities are now available, their structural integration into spatially interlinked prototissues that communicate and display coordinated functions remains a considerable challenge. Here we describe the programmed assembly of synthetic prototissue constructs based on the bio-orthogonal adhesion of a spatially confined binary community of protein-polymer protocells, termed proteinosomes. The thermoresponsive properties of the interlinked proteinosomes are used collectively to generate prototissue spheroids capable of reversible contractions that can be enzymatically modulated and exploited for mechanochemical transduction. Overall, our methodology opens up a route to the fabrication of artificial tissue-like materials capable of collective behaviours, and addresses important emerging challenges in bottom-up synthetic biology and bioinspired engineering.

Modulation of Higher-order Behaviour in Model Protocell Communities by Artificial Phagocytosis.

Collective behaviour in mixed populations of synthetic protocells is an unexplored area of bottom-up synthetic biology. The dynamics of a model protocell community is exploited to modulate the function and higher-order behaviour of mixed populations of bioinorganic protocells in response to a process of artificial phagocytosis. Enzyme-loaded silica colloidosomes are spontaneously engulfed by magnetic Pickering emulsion (MPE) droplets containing complementary enzyme substrates to initiate a range of processes within the host/guest protocells. Specifically, catalase, lipase, or alkaline phosphatase-filled colloidosomes are used to trigger phagocytosis-induced buoyancy, membrane reconstruction, or hydrogelation, respectively, within the MPE droplets. The results highlight the potential for exploiting surface-contact interactions between different membrane-bounded droplets to transfer and co-locate discrete chemical packages (artificial organelles) in communities of synthetic protocells.

Design and construction of higher-order structure and function in proteinosome-based protocells.

The design and construction of higher-order structure and function in proteinosome microcompartments enclosed by a cross-linked membrane of amphiphilic bovine serum albumin/poly(N-isopropylacrylamide) (BSA-NH2/PNIPAAm) nanoconjugates is described. Three structure/function relationships are investigated: (i) differential chemical cross-linking for the control of membrane disassembly and regulated release of encapsulated genetic polymers; (ii) enzyme-mediated hydrogel structuring of the internal microenvironment to increase mechanical robustness and generate a molecularly crowded reaction environment; and (iii) self-production of a membrane-enclosing outer hydrogel wall for generating protease-resistant forms of the protein-polymer protocells. Our results highlight the potential of integrating aspects of supramolecular and polymer chemistry into the design and construction of novel bioinspired microcompartments as a step toward small-scale materials systems based on synthetic cellularity.

Spontaneous structuration in coacervate-based protocells by polyoxometalate-mediated membrane assembly.

Molecularly crowded, polyelectrolyte/ribonucleotide-enriched membrane-free coacervate droplets are transformed into membrane-bounded sub-divided vesicles by using a polyoxometalate-mediated surface-templating procedure. The coacervate to vesicle transition results in reconstruction of the coacervate micro-droplets into novel three-tiered micro-compartments comprising a semi-permeable negatively charged polyoxometalate/polyelectrolyte outer membrane, a sub-membrane coacervate shell, and an internal aqueous lumen. We demonstrate that organic dyes, ssDNA, magnetic nanoparticles and enzymes can be concentrated into the interior of the micro-compartments by sequestration into the coacervate micro-droplets prior to vesicle formation. The vesicle-encapsulated proteins are inaccessible to proteases in the external medium, and can be exploited for the spatial localization and coupling of two-enzyme cascade reactions within single or between multiple populations of hybrid vesicles dispersed in aqueous media.

Artificial cytoskeletal structures within enzymatically active bio-inorganic protocells.

The fabrication of enzymatically active, semi-permeable bio-inorganic protocells capable of self-assembling a cytoskeletal-like interior and undergoing small-molecule dephosphorylation reactions is described. Reversible disassembly of an amino acid-derived supramolecular hydrogel within the internalized reaction space is used to tune the enzymatic activity of the nanoparticle-bounded inorganic compartments.

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-ß. 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.

Chemical-mediated translocation in protocell-based microactuators.

Artificial cell-like communities participate in diverse modes of chemical interaction but exhibit minimal interfacing with their local environment. Here we develop an interactive microsystem based on the immobilization of a population of enzyme-active semipermeable proteinosomes within a helical hydrogel filament to implement signal-induced movement. We attach large single-polynucleotide/peptide microcapsules at one or both ends of the helical protocell filament to produce free-standing soft microactuators that sense and process chemical signals to perform mechanical work. Different modes of translocation are achieved by synergistic or antagonistic enzyme reactions located within the helical connector or inside the attached microcapsule loads. Mounting the microactuators on a ratchet-like surface produces a directional push-pull movement. Our methodology opens up a route to protocell-based chemical systems capable of utilizing mechanical work and provides a step towards the engineering of soft microscale objects with increased levels of operational autonomy.

Catalytic processing in ruthenium-based polyoxometalate coacervate protocells.

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 micro-droplets 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 multi-catalytic protocell communities. Our results highlight a new type of catalytic micro-compartment with multi-functional activity and provide a step towards the development of protocell reaction networks.

Artificial morphogen-mediated differentiation in synthetic protocells.

The design and assembly of artificial protocell consortia displaying dynamical behaviours and systems-based properties are emerging challenges in bottom-up synthetic biology. Cellular processes such as morphogenesis and differentiation rely in part on reaction-diffusion gradients, and the ability to mimic rudimentary aspects of these non-equilibrium processes in communities of artificial cells could provide a step to life-like systems capable of complex spatiotemporal transformations. Here we expose acoustically formed arrays of initially identical coacervate micro-droplets to uni-directional or counter-directional reaction-diffusion gradients of artificial morphogens to induce morphological differentiation and spatial patterning in single populations of model protocells. Dynamic reconfiguration of the droplets in the morphogen gradients produces a diversity of membrane-bounded vesicles that are spontaneously segregated into multimodal populations with differentiated enzyme activities. Our results highlight the opportunities for constructing protocell arrays with graded structure and functionality and provide a step towards the development of artificial cell platforms capable of multiple operations.

Chitin and carbon nanotube composites as biocompatible scaffolds for neuron growth.

The design of biocompatible implants for neuron repair/regeneration ideally requires high cell adhesion as well as good electrical conductivity. Here, we have shown that plasma-treated chitin carbon nanotube composite scaffolds show very good neuron adhesion as well as support of synaptic function of neurons. The addition of carbon nanotubes to a chitin biopolymer improved the electrical conductivity and the assisted oxygen plasma treatment introduced more oxygen species onto the chitin nanotube scaffold surface. Neuron viability experiments showed excellent neuron attachment onto plasma-treated chitin nanotube composite scaffolds. The support of synaptic function was evident on chitin/nanotube composites, as confirmed by PSD-95 staining. The biocompatible and electrically-conducting chitin nanotube composite scaffold prepared in this study can be used for in vitro tissue engineering of neurons and, potentially, as an implantable electrode for stimulation and repair of neurons.

Integrative self-assembly of functional hybrid nanoconstructs by inorganic wrapping of single biomolecules, biomolecule arrays and organic supramolecular assemblies.

Synthesis of functional hybrid nanoscale objects has been a core focus of the rapidly progressing field of nanomaterials science. In particular, there has been significant interest in the integration of evolutionally optimized biological systems such as proteins, DNA, virus particles and cells with functional inorganic building blocks to construct mesoscopic architectures and nanostructured materials. However, in many cases the fragile nature of the biomolecules seriously constrains their potential applications. As a consequence, there is an on-going quest for the development of novel strategies to modulate the thermal and chemical stabilities, and performance of biomolecules under adverse conditions. This feature article highlights new methods of "inorganic molecular wrapping" of single or multiple protein molecules, individual double-stranded DNA helices, lipid bilayer vesicles and self-assembled organic dye superstructures using inorganic building blocks to produce bio-inorganic nanoconstructs with core-shell type structures. We show that spatial isolation of the functional biological nanostructures as "armour-plated" enzyme molecules or polynucleotide strands not only maintains their intact structure and biochemical properties, but also enables the fabrication of novel hybrid nanomaterials for potential applications in diverse areas of bionanotechnology.

Enhanced catalytic activity in organic solvents using molecularly dispersed haemoglobin-polymer surfactant constructs.

The surface of haemoglobin (Hb) is chemically modified to produce molecular dispersions of discrete core-shell Hb-polymer surfactant bionanoconjugates in water and organic solvents. The hybrid nanoconstructs exhibit peroxidase-like catalytic activity with enhanced turnover rates compared with native Hb in water.

Interfacial assembly of protein-polymer nano-conjugates into stimulus-responsive biomimetic protocells.

The mechanism of spontaneous assembly of microscale compartments is a central question for the origin of life, and has technological repercussions in diverse areas such as materials science, catalysis, biotechnology and biomedicine. Such compartments need to be semi-permeable, structurally robust and capable of housing assemblages of functional components for internalized chemical transformations. In principle, proteins should be ideal building blocks for the construction of membrane-bound compartments but protein vesicles with cell-like properties are extremely rare. Here we present an approach to the interfacial assembly of protein-based micro-compartments (proteinosomes) that are delineated by a semi-permeable, stimulus-responsive, enzymatically active, elastic membrane consisting of a closely packed monolayer of conjugated protein-polymer building blocks. The proteinosomes can be dispersed in oil or water, thermally cycled to temperatures of 70 °C, and partially dried and re-inflated without loss of structural integrity. As a consequence, they exhibit protocellular properties such as guest molecule encapsulation, selective permeability, gene-directed protein synthesis and membrane-gated internalized enzyme catalysis.

A new general synthetic strategy for phase-pure complex functional materials.

The ability of ionic liquids to solvate inorganic salts completely has to date never been employed in the synthesis of complex inorganic materials. Here, we demonstrate that complex functional oxides, even those traditionally considered extremely difficult to synthesize in bulk, such as quinternary superconductors, are produced with no impurity phases and on timescales that are much shorter than other synthetic techniques.

Fabrication of polypyrrole nano-arrays in lysozyme single crystals.

A template-directed method for the synthesis and organization of partially oxidized polypyrrole (PPy) nanoscale arrays within the solvent channels of glutaraldehyde-cross-linked lysozyme single crystals is presented. Macroscopic single crystals of the periodically arranged protein-polymer superstructure are electrically conductive, insoluble in water and organic solvents, and display increased levels of mechanical plasticity compared with native cross-linked lysozyme crystals.

Fabrication of functional bioinorganic nanoconstructs by polymer-silica wrapping of individual myoglobin molecules.

Discrete core-shell hybrid nanoparticles comprising individual met-myoglobin (met-Mb) molecules incarcerated within an ultrathin polymer/silica shell were prepared without loss of biofunctionality by a facile self-assembly procedure. Solubilisation of met-Mb in cyclohexane in the near-absence of water was achieved by wrapping individual protein molecules in the amphiphilic triblock copolymer poly(ethylene-oxide)19-poly(propylene-oxide)69-poly(ethylene-oxide)19 (EO19-PO69-EO19, P123). Addition of tetramethoxysilane to the met-Mb/P123 conjugates in cyclohexane produced discrete nanoparticles that contained protein, polymer and silica, and which were 3-5.5 nm in size, consistent with the entrapment of single molecules of met-Mb. The hybrid nanoconstructs were isolated and re-dispersed in water without loss of secondary structure, and remained functionally active with respect to redox reactions and CO and O2 ligand binding at the porphyrin metallocentre. The incarcerated met-Mb biomolecules showed enhanced thermal stability up to a temperature of around 85 °C. These properties, along with the high biocompatibility of silica and P123, suggest that the silicified protein-polymer constructs could be utilised as functional nanoscale components in bionanotechnology.

Immobilisation and encapsulation of functional protein-inorganic constructs.

Self-assembly methods for the immobilisation or encapsulation of the positively charged redox protein, cytochrome c (cyt c), in layered organoclays or silica nanoparticles, respectively, are described and contrasted. Protein-polymer-organoclay nanocomposites are produced by spontaneous restacking of delaminated aminopropyl-functionalised magnesium phyllosilicate sheets in the presence of an aqueous solution of poly(sodium 4-styrene sulfonate) (PSS) and cyt c. In contrast, single molecules of cyt c are encapsulated in silica nanoparticles by sol-gel reactions at the oil-water interface of microemulsion water droplets. In both cases, the protein molecules remain structurally intact after entrapment, are accessible to small molecule redox agents, exhibit excellent peroxidase activity in the presence of hydrogen peroxide, and show enhanced stability and catalytic properties under adverse conditions of pH. The ability to prepare functional protein-inorganic conjugates in general could significantly extend the technological scope of biological products and processes, and should therefore be an important adjunct in the translation of synthetic biology to real-life applications.

Fabrication of mineralized polymeric nanofibrous composites for bone graft materials.

Poly-L-lactic acid (PLLA) and PLLA/collagen (50% PLLA+50% collagen; PLLA/Col) nanofibers were fabricated using electrospinning. Mineralization of these nanofibers was processed using a modified alternating soaking method. The structural properties and morphologies of mineralized PLLA and PLLA/Col nanofibers were investigated using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and contact angle measurements. Human bone-derived osteoblasts were cultured on the materials for up to 1 week to assess the biological properties of the nanofibrous composites. Cell attachment on these nanocomposites was also tested within 1 h of culture at room temperature. The mechanical properties of the cell-nanocomposite constructs were determined using tensile testing. From our results, the bone-like nano-hydroxyapatite (n-HA) was successfully deposited on the PLLA and PLLA/Col nanofibers. We observed that the formation of n-HA on PLLA/Col nanofibers was faster and significantly more uniform than on pure PLLA nanofibers. The n-HA significantly improved the hydrophilicity of PLLA/Col nanofibers. From the results of cell attachment studies, n-HA deposition enhanced the cell capture efficacy at the 20-minute time point for PLLA nanofibers. The E-modulus values for PLLA+n-HA with cells (day 1 and day 4) were significantly higher than for PLLA+n-HA without cells. Based on these observations, we have demonstrated that n-HA deposition on nanofibers is a promising strategy for early cell capture.