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A rationally designed dual purpose non-canonical amino acid (Trz) has been synthesised and successfully incorporated into a protein scaffold via genetic code expansion. Trz contains a 5-pyridyl-1,2,4-triazine system, which allows for inverse electron-demand Diels-Alder (IEDDA) reactions to occur on the triazine ring and for metal ions to be chelated both before and after the click reaction. Trz was successfully incorporated into a protein scaffold and the IEDDA utility of Trz demonstrated through the site-specific labelling of the purified protein with a bicyclononyne. Additionally, Trz was shown to successfully coordinate a cyclometallated iridium(III) centre, providing access to a bioorthogonal luminogenic probe. The luminescent properties of the Ir(III)-bound protein blue-shift upon IEDDA click reaction with bicyclononyne, providing a unique method for monitoring the extent and location of the labelling reaction. In summary, Trz is a new dual purpose non-canonical amino acid, which has great potential for myriad bioapplications where metal-based functionality is required, for example in imaging, catalysis, or photo-dynamic therapy, in conjunction with a bioorthogonal reactive handle to impart additional functionalities, such as dual modality imaging or therapeutic payloads.

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Most organophosphates (OPs) are hydrophobic, and after exposure, can sequester into lipophilic regions within the body, such as adipose tissue, resulting in long term chronic effects. Consequently, there is an urgent need for therapeutic agents that can decontaminate OPs in these hydrophobic regions. Accordingly, an enzyme-polymer surfactant nanocomplex is designed and tested comprising chemically supercharged phosphotriesterase (Agrobacterium radiobacter; arPTE) electrostatically conjugated to amphiphilic polymer surfactant chains ([cat.arPTE][S-]). Experimentally-derived structural data are combined with molecular dynamics (MD) simulations to provide atomic level detail on conformational ensembles of the nanocomplex using dielectric constants relevant to aqueous and lipidic microenvironments. These show the formation of a compact admicelle pseudophase surfactant corona under aqueous conditions, which reconfigures to yield an extended conformation at a low dielectric constant, providing insight into the mechanism underpinning cell membrane binding. Significantly, it demonstrated that [cat.arPTE][S-] spontaneously binds to human mesenchymal stem cell membranes (hMSCs), resulting in on-cell OP hydrolysis. Moreover, the nanoconstruct can endocytose and partition into the intracellular fatty vacuoles of adipocytes and hydrolyze sequestered OP.

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Correction for 'Flax fibre reinforced alginate poloxamer hydrogel: assessment of mechanical and 4D printing potential' by Charles de Kergariou et al., Soft Matter, 2024, 20, 4021-4034, https://doi.org/10.1039/D4SM00135D.

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The mechanical and printing performance of a new biomaterial, flax fibre-reinforced alginate-poloxamer based hydrogel, for load-bearing and 4D printing biomedical applications is described in this study. The-self suspendable ability of the material was evaluated by optimising the printing parameters and conducting a collapse test. 1% of the flax fibre weight fraction was sufficient to obtain an optimum hydrogel composite from a mechanical perspective. The collapse test showed that the addition of flax fibres allowed a consistent print without support over longer distances (8 and 10 mm) than the unreinforced hydrogel. The addition of 1% of flax fibres increased the viscosity by 39% and 129% at strain rates of 1 rad s-1 and 5 rad s-1, respectively, compared to the unreinforced hydrogel. The distributions of fibre size and orientation inside the material were also evaluated to identify the internal morphology of the material. The difference of coefficients of moisture expansion between the printing direction (1.29 × 10-1) and the transverse direction (6.03 × 10-1) showed potential for hygromorphic actuation in 4D printing. The actuation authority was demonstrated by printing a [0°; 90°] stacking sequence and rosette-like structures, which were then actuated using humidity gradients. Adding fibres to the hydrogel improved the repeatability of the actuation, while lowering the actuation authority from 0.11 mm-1 to 0.08 mm-1. Overall, this study highlighted the structural and actuation-related benefits of adding flax fibres to hydrogels.

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Catalytically active materials for the enhancement of personalized protective equipment (PPE) could be advantageous to help alleviate threats posed by neurotoxic organophosphorus compounds (OPs). Accordingly, a chimeric protein comprised of a supercharged green fluorescent protein (scGFP) and phosphotriesterase from Agrobacterium radiobacter (arPTE) was designed to drive the polymer surfactant (S-)-mediated self-assembly of microclusters to produce robust, enzymatically active materials. The chimera scGFP-arPTE was structurally characterized via circular dichroism spectroscopy and synchrotron radiation small-angle X-ray scattering, and its biophysical properties were determined. Significantly, the chimera exhibited greater thermal stability than the native constituent proteins, as well as a higher catalytic turnover number (kcat). Furthermore, scGFP-arPTE was electrostatically complexed with monomeric S-, driving self-assembly into [scGFP-arPTE][S-] nanoclusters, which could be dehydrated and cross-linked to yield enzymatically active [scGFP-arPTE][S-] porous films with a high-order structure. Moreover, these clusters could self-assemble within cotton fibers to generate active composite textiles without the need for the pretreatment of the fabrics. Significantly, the resulting materials maintained the biophysical activities of both constituent proteins and displayed recyclable and persistent activity against the nerve agent simulant paraoxon.

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One of the major challenges within the emerging field of injectable stem cell therapies for articular cartilage (AC) repair is the retention of sufficient viable cell numbers at the site of injury. Even when delivered via intra-articular injection, the number of stem cells retained at the target is often low and declines rapidly over time. To address this challenge, an artificial plasma membrane binding nanocomplex was rationally designed to provide human mesenchymal stem cells (hMSCs) with increased adhesion to articular cartilage tissue. The nanocomplex comprises the extracellular matrix (ECM) binding peptide of a placenta growth factor-2 (PlGF-2) fused to a supercharged green fluorescent protein (scGFP), which was electrostatically conjugated to anionic polymer surfactant chains to yield [S-]scGFP_PlGF2. The [S-]scGFP_PlGF2 nanocomplex spontaneously inserts into the plasma membrane of hMSCs, is not cytotoxic, and does not inhibit differentiation. The nanocomplex-modified hMSCs showed a significant increase in affinity for immobilised collagen II, a key ECM protein of cartilage, in both static and dynamic cell adhesion assays. Moreover, the cells adhered strongly to bovine ex vivo articular cartilage explants resulting in high cell numbers. These findings suggest that the re-engineering of hMSC membranes with [S-]scGFP_PlGF2 could improve the efficacy of injectable stem cell-based therapies for the treatment of damaged articular cartilage.

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Here, we describe a facile route to the synthesis of enzymatically active highly fabricable plastics, where the enzyme is an intrinsic component of the material. This is facilitated by the formation of an electrostatically stabilized enzyme-polymer surfactant nanoconstruct, which, after lyophilization and melting, affords stable macromolecular dispersions in a wide range of organic solvents. A selection of plastics can then be co-dissolved in the dispersions, which provides a route to bespoke 3D enzyme plastic nanocomposite structures using a wide range of fabrication techniques, including melt electrowriting, casting, and piston-driven 3D printing. The resulting constructs comprising active phosphotriesterase (arPTE) readily detoxify organophosphates with persistent activity over repeated cycles and for long time periods. Moreover, we show that the protein guest molecules, such as arPTE or sfGFP, increase the compressive Young's modulus of the plastics and that the identity of the biomolecule influences the nanomorphology and mechanical properties of the resulting materials. Overall, we demonstrate that these biologically active nanocomposite plastics are compatible with state-of-the-art 3D fabrication techniques and that the methodology could be readily applied to produce robust and on-demand smart nanomaterial structures.

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We present a new methodology for the generation of discrete molecularly dispersed enzyme-polymer-surfactant bioconjugates. Significantly, we demonstrate that >3-fold increase in the catalytic efficiency of the diffusion-limited phosphotriesterase arPTE can be achieved through sequential electrostatic addition of cationic and anionic polymer surfactants, respectively. Here, the polymer surfactants assemble on the surface of the enzyme via ion exchange to yield a compact corona. The observed rate enhancement is consistent with a mechanism whereby the polymer-surfactant corona gives rise to a decrease in the dielectric constant in the vicinity of the active site of the enzyme, accelerating the rate-determining product diffusion step. The facile methodology has significant potential for increasing the efficiency of enzymes and could therefore have a substantially positive impact for industrial enzymology.

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