Dendrimersome Synthetic Cells Harbor Cell Division Machinery of Bacteria

The integration of active cell machinery with synthetic building blocks is the bridge toward developing synthetic cells with biological functions and beyond. Self-replication is one of the most important tasks of living systems, and various complex machineries exist to execute it. In Escherichia coli, a contractile division ring is positioned to mid-cell by concentration oscillations of self-organizing proteins (MinCDE), where it severs membrane and cell wall. So far, the reconstitution of any cell division machinery has exclusively been tied to liposomes. Here, the reconstitution of a rudimentary bacterial divisome in fully synthetic bicomponent dendrimersomes is shown. By tuning the membrane composition, the interaction of biological machinery with synthetic membranes can be tailored to reproduce its dynamic behavior. This constitutes an important breakthrough in the assembly of synthetic cells with biological elements, as tuning of membrane-divisome interactions is the key to engineering emergent biological behavior from the bottom-up

Phosphorylation disrupts long-distance electron transport in cytochrome c

It has been recently shown that electron transfer between mitochondrial cytochrome c and the cytochrome c1 subunit of the cytochrome bc1 can proceed at long-distance through the aqueous solution. Cytochrome c is thought to adjust its activity by changing the affinity for its partners via Tyr48 phosphorylation, but it is unknown how it impacts the nanoscopic environment, interaction forces, and long-range electron transfer. Here, we constrain the orientation and separation between cytochrome c1 and cytochrome c or the phosphomimetic Y48pCMF cytochrome c, and deploy an array of single-molecule, bulk, and computational methods to investigate the molecular mechanism of electron transfer regulation by cytochrome c phosphorylation. We demonstrate that phosphorylation impairs long-range electron transfer, shortens the long-distance charge conduit between the partners, strengthens their interaction, and departs it from equilibrium. These results unveil a nanoscopic view of the interaction between redox protein partners in electron transport chains and its mechanisms of regulation

Highly versatile polyelectrolyte complexes for improving the enzyme replacement therapy of lysosomal storage disorders

Lysosomal storage disorders are currently treated by enzyme replacement therapy (ERT) through the direct administration of the unprotected recombinant protein to the patients. Herein we present an ionically crosslinked polyelectrolyte complex (PEC) composed of trimethyl chitosan (TMC) and -galactosidase A (GLA), the defective enzyme in Fabry disease, with the capability of directly targeting endothelial cells by incorporating peptide ligands containing the RGD sequence. We assessed the physicochemical properties, cytotoxicity and hemocompatibility of RGD-targeted and un-targeted PECs, the uptake by endothelial cells and the intracellular activity of PECs in cell culture models of Fabry disease. Moreover, we also explored the effect of different freezedrying procedures in the overall activity of the PECs. Our results indicate that the use of integrin-binding RGD moiety within the PEC increases their uptake and the efficacy of the GLA enzyme, while the freeze-drying allows keeping intact the activity of the therapeutic protein. Overall, these results highlight the potential of TMC-based PECs as a highly versatile and feasible drug delivery system for improving the ERT of lysosomal storage disorders

Hierarchical Quatsome-RGD Nanoarchitectonic Surfaces for Enhanced Integrin-Mediated Cell Adhesion

The synthesis and study of the tripeptide Arg-Gly-Asp (RGD), the binding site of different extracellular matrix proteins, e.g., fibronectin and vitronectin, has allowed the production of a wide range of cell adhesive surfaces. Although the surface density and spacing of the RGD peptide at the nanoscale have already shown a significant influence on cell adhesion, the impact of its hierarchical nanostructure is still rather unexplored. Accordingly, a versatile colloidal system named quatsomes, based on fluid nanovesicles formed by the self-assembling of cholesterol and surfactant molecules, has been devised as a novel template to achieve hierarchical nanostructures of the RGD peptide. To this end, RGD was anchored on the vesicle's fluid membrane of quatsomes, and the RGD-functionalized nanovesicles were covalently anchored to planar gold surfaces, forming a state of quasi-suspension, through a long poly(ethylene glycol) (PEG) chain with a thiol termination. An underlying self-assembled monolayer (SAM) of a shorter PEG was introduced for vesicle stabilization and to avoid unspecific cell adhesion. In comparison with substrates featuring a homogeneous distribution of RGD peptides, the resulting hierarchical nanoarchitectonic dramatically enhanced cell adhesion, despite lower overall RGD molecules on the surface. The new versatile platform was thoroughly characterized using a multitechnique approach, proving its enhanced performance. These findings open new methods for the hierarchical immobilization of biomolecules on surfaces using quatsomes as a robust and novel tissue engineering strategy.This work was supported by MICINN (PID2019-105622RBI00, MAT2016-80826-R, PID2019-111682RB-I00, PID2020-115296RA-I00, CTQ2015-66194-R; SAF2014-60138-R, RTI2018-093831-B-I00, and PDC2021-121481-I00); Instituto de Salud Carlos III (ISCIII) through the Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN (FlexQS-skin, FlexCAB, BBN18PI01, BBN20PIV02, and CB/06/0074); Generalitat de Catalunya (grants 2017-SGR-918, 2017-SGR-229, 2017-SGR-1442, 2017-SGR-1439); the Fundació Marató de TV3 (Nr. 201812); the COST Action CA15126 Between Atom and Cell, and “ERDF A way of making Europe”. J.G. acknowledges financial support from the Ramón y Cajal Program (RYC-2017-22614) from MICINN and the Max Planck Society through the Max Planck Partner Group “Dynamic Biomimetics for Cancer Immunotherapy” in collaboration with the Max Planck Institute for Medical Research (Heidelberg, Germany). This work has received funding from the European Union’s Horizon 2020 research and innovation program through grant agreements 953110 (PHOENIX), 720942 (Smart4Fabry), 101007804 (MICRO4NANO), and 801342 (granted to the Agency for Business Competitiveness ACCIÓ through a Tecniospring Industry fellowship (TECSPR19-1-0065)). ICMAB acknowledges support from MICINN through the “‘Severo Ochoa”’ Programme for Centres of Excellence in R&D (CEX2019-000917-S). J.M. acknowledges a “Juan de la Cierva” fellowship from MICINN. J.T-M. acknowledges an FI-AGAUR grant (2020FI_B2 00137) from Generalitat de Catalunya and the European Social Fund. We also acknowledge the ICTS “NANBIOSIS for the support of the Synthesis of Peptides Unit (U3) at IQAC–CSIC (https://www.nanbiosis.es/portfolio/u3-synthesis-of-peptides-unit/) and the Biomaterial Processing and Nanostructuring Unit (U6) at ICMAB-CSIC (https://www.nanbiosis.es/portfolio/u6-biomaterial-processing-and-nanostructuring-unit/). We are grateful to the SMP unit of the Scientific and Technological Centers of University of Barcelona (CCiTUB). This work has been developed under the “Biochemistry, Molecular Biology and Biomedicine” and “Materials Science” Ph.D. programs of Universitat Autònoma de Barcelona (UAB).With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe

Polymer-based drug delivery systems under investigation for enzyme replacement and other therapies of lysosomal storage disorders

Lysosomes play a central role in cellular homeostasis and alterations in this compartment associate with many diseases. The most studied example is that of lysosomal storage disorders (LSDs), a group of 60 + maladies due to genetic mutations affecting lysosomal components, mostly enzymes. This leads to aberrant intracellular storage of macromolecules, altering normal cell function and causing multiorgan syndromes, often fatal within the first years of life. Several treatment modalities are available for a dozen LSDs, mostly consisting of enzyme replacement therapy (ERT) strategies. Yet, poor biodistribution to main targets such as the central nervous system, musculoskeletal tissue, and others, as well as generation of blocking antibodies and adverse effects hinder effective LSD treatment. Drug delivery systems are being studied to surmount these obstacles, including polymeric constructs and nanoparticles that consti-tute the focus of this article. We provide an overview of the formulations being tested, the diseases they aim to treat, and the results observed from respective in vitro and in vivo studies. We also discuss the advantages and disadvantages of these strategies, the remaining gaps of knowledge regarding their per-formance, and important items to consider for their clinical translation. Overall, polymeric nanocon-structs hold considerable promise to advance treatment for LSDs.(c) 2023 Elsevier B.V. All rights reserved

Interrogation of Single Synthetic Polymer Chains and Polysaccharides by AFM-Based Force Spectroscopy

This contribution reviews selected mechanical experiments on individual flexible macromolecules using single-molecule force spectroscopy (SMFS) based on atomic force microscopy. Focus is placed on the analysis of elasticity and conformational changes in single polymer chains upon variation of the external environment, as well as on conformational changes induced by the mechanical stress applied to individual macromolecular chains. Various experimental strategies regarding single-molecule manipulation and SMFS testing are discussed, as is theoretical analysis through single-chain elasticity models derived from statistical mechanics. Moreover, a complete record, reported to date, of the parameters obtained when applying the models to fit experimental results on synthetic polymers and polysaccharides is presented.\ud \u

Force Spectroscopy of Hyaluronan by Atomic Force Microscopy: From Hydrogen-Bonded Networks toward Single-Chain Behavior\ud

The conformational behavior of hyaluronan (HA) polysaccharide chains in aqueous NaCl solution was characterized directly at the single-molecule level. This comunication reports on one of the first single-chain atomic force microscopy (AFM) experiments performed at variable temperatures, investigating the influence of the temperature on the stability of the HA single-chain conformation. Through AFM single-molecule force spectroscopy, the temperature destabilization of a local structure was proven. This structure involved a hydrogen-bonded network along the polymeric chain, with hydrogen bonds between the polar groups of HA and possibly water, and a change from a nonrandom coil to a random coil behavior was observed when increasing the temperature from 29 ± 1 to 46 ± 1 °C. As a result of the applied force, this superstructure was found to break progressively at room temperature. The use of a hydrogen-bonding breaker solvent demonstrated the hydrogen-bonded water-bridged nature of the network structure of HA single chains in aqueous NaCl solution. \u

Morphology profiles obtained by reaction-induced phase separation in epoxy/polysulfone/poly(ether imide) systems

The reaction-induced phase separation in epoxy/aromatic diamine formulations simultaneously modified with two immiscible thermoplastics (TPs), poly(ether imide) (PEI) and polysulfone (PSF), has been studied. The epoxy monomer was based on the diglycidyl ether of bisphenol A (DGEBA) and the aromatic diamine was 4,4′-methylenebis(3-chloro 2,6-diethylaniline) (MCDEA). Phase-separation conversions are reported for various PSF/PEI proportions for blends containing 10 wt% total TP. On the basis of phase-separation results, a conversion–composition phase diagram at 200 °C was compiled. This diagram was used to design particular cure cycles in order to generate different morphologies during the phase-separation process. It was found that, depending on the PSF/PEI ratio employed, a particulate or a morphology characterized by a distribution of irregular PEI-rich domains dispersed in an epoxy-rich phase was obtained for initially miscible blends. Scanning electron microscopy (SEM) characterization revealed that the PEI-rich phase exhibits a phase-inverted structure and the epoxy-rich matrix presents a bimodal size distribution of TP-rich particles. For PSF/PEI ratios near the miscibility limit, slight temperature change result in morphology profiles.Fil: Giannotti, Marina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; ArgentinaFil: Mondragon, I.. Universidad del País Vasco; EspañaFil: Galante, Maria Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; ArgentinaFil: Oyanguren, Patricia Angelica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; Argentin

The protein matrix of plastocyanin supports long-distance charge transport with photosystem I and the copper ion regulates its spatial span and conductance

Charge exchange is the fundamental process that sustains cellular respiration and photosynthesis by shuttling electrons in a cascade of electron transfer (ET) steps between redox cofactors. While intraprotein charge exchange is well characterized in protein complexes bearing multiple redox sites, interprotein processes are less understood due to the lack of suitable experimental approaches and the dynamic nature of the interactions. Proteins constrained between electrodes are known to support electron transport (ETp) through the protein matrix even without redox cofactors, as the charges housed by redox sites in ET are furnished by the electrodes in ETp configuration. However, it is unknown whether protein ETp mechanisms apply to the interprotein medium that is present in physiological conditions. Here, we study interprotein charge exchange between plant photosystem I (PSI) and its soluble redox partner plastocyanin (Pc) and address the role of the Pc copper center. Using electrochemical scanning tunnelling spectroscopy (ECSTS) current-distance and blinking measurements we respectively quantify the spatial span of charge exchange between individual Pc/PSI pairs and ETp through transient Pc/PSI complexes. Pc devoid of the redox center (Pcapo) can exchange charge with PSI and it does so at longer distances than with the copper ion (Pcholo). Conductance bursts associated to Pcapo/PSI complex formation are higher than in Pcholo/PSI. Thus, copper ions are not required for long distance ETp between PSI and Pc but regulate its spatial span and conductance. Our results suggest that the redox center that carries the charge in Pc is not necessary to exchange it in interprotein ET through the aqueous solution, and question the canonical view of tight complex binding between redox protein partners