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This study was supported by projects RTI-2018-095794-A-C22, RYC-2016-21042 and project PID2020-118498GB-I00 funded by MCIN/AEI/10.13039/501100011033 and projects P18-FR-3533 and P18-TP-0969 by FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades (Spain). Thanks go to the CIC personnel of the University of Granada for technical assistance. MCMT acknowledges grant PRE2018-083773 funded by MCIN/AEI/10.13039/501100011033 and FSE "El FSE invierte en tu futuro", Spain. GBRR also acknowledges Junta de Andalucia for her postdoctoral contract within the PAIDI 2020 program ( DOC_01383)., Self-assembled peptides and proteins have turned out to be excellent templates for the growth of inorganic minerals and can be used to emulate natural biomineralization processes. Doing this, researchers have developed complex sophisticated materials with properties, in some cases, similar to those found in nature. Of special interest is the development of scaffolds able to guide bone regeneration. The bone tissue comprises an organic matrix composed of aligned collagen fibers containing nanoapatite crystals oriented along the fiber direction. During bone mineralization, both processes, the self-assembly of collagen fibrils and mineralization occur simultaneously. Collagen fibers are able to control calcium phosphate nucleation and subsequent apatite crystal growth at a very limited range of collagen density and ionic concentration. In this study, we reproduced the simultaneity of both processes using an artificial peptide fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) that has the ability to self-assemble in water after the addition of Ca2+ ions. Therefore, the peptide self-assembly process and the mineralization of apatite are Ca-demanding processes and occur simultaneously. The role of peptide and ionic concentrations has been investigated affording organic/inorganic hybrid hydrogels with different degrees of homogeneity and mineralization. Interestingly, at very low Ca2+ concentrations, we found that apatite nanocrystals are integrated into Fmoc-FF fibrils and oriented as in biologically mineralized collagen fibrils, the basic building blocks of bone., FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades (Spain) P18-FR-3533 P18-TP-0969, FSE "El FSE invierte en tu futuro", Spain, Junta de Andalucia DOC_01383; RTI-2018-095794-A-C22; RYC-2016-21042; PID2020-118498GB-I00; MCIN/AEI/10.13039/501100011033; PRE2018-083773

This study was supported by projects CTQ2017-86568-R and FIS2017-85954-R (Ministerio de Economia, Industria y Competitividad, MINECO, and Agencia Estatal de Investigacion, AEI, Spain, cofunded by Fondo Europeo de Desarrollo Regional, FEDER, European Union), and by FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades (Spain) project P18-FR-3533. We want to thank "Unidad de Excelencia Quimica aplicada a Biomedicina y Medioambiente" of the University of Granada. Thanks go to the CIC personnel of the University of Granada for technical assistance. We thank the Centro de Servicios de Informatica y Redes de Comunicaciones (CSIRC), Universidad de Granada, for providing the computing time. Authors acknowledge to Dr Rosario Herranz and Dr Francisco Fueyo-Gonzalez for the fluorophore 9-azetidinyl-5-butyl-quinolimide (AQui) used in this study, which was synthesized at Instituto de Quimica Medica-CSIC (IQM-CSIC) with the support of the Ministerio de Ciencia e Innovacion/Agencia Estatal de Investigacion grant FU2015-67284-R., Making use of the combination of multiparametric Fluorescence Lifetime Imaging Microscopy (FLIM) and single-molecule Fluorescence Lifetime Correlation Spectroscopy (FLCS), we have been able to study for the early stages of the fluorenylmethyloxycarbonyl-diphenylalanine (Fmoc-FF) self-assembly process with single-molecule resolution, the kinetics of fiber formation, the packaging of the peptides within the fibers and the capacity of the peptides to reassemble after disruption (self-healing) in the presence of different metallic cations. Other techniques such as FTIR, TEM, DSC and DFT calculations support our findings. The impact that the mechanism of self-assembly has on the physical (rigidity and self-healing) properties of the resulting gels have also been evaluated by rheology. Calcium ions are able to promote the self-assembly of Fmoc-FF faster and more efficiently, forming more rigid hydrogels than do cesium ions. The reasons behind this effect may be explained by the different capacities that these two cations have to coordinate with the peptide, modulate its hydrophobicity and stabilize the water-solute interphase. These findings shed light on the impact that small changes have on the process of self-assembly and can help to understand the influence of the environmental conditions on the in vivo uncontrolled self-assembly of certain proteins., Ministerio de Economia, Industria y Competitividad, MINECO CTQ2017-86568-R FIS2017-85954-R, Agencia Estatal de Investigacion, AEI, Spain CTQ2017-86568-R FIS2017-85954-R, Fondo Europeo de Desarrollo Regional, FEDER, European Union CTQ2017-86568-R FIS2017-85954-R, FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades (Spain) P18-FR-3533, Ministerio de Ciencia e Innovacion/Agencia Estatal de Investigacion FU2015-67284-R

Purpose: New antiviral agents were prepared by attaching derivatives of sialic acid (1) and of the drug zanamivir (2) to poly(isobutylene-alt-maleic anhydride) (poly-(1 + 2)) or by mixing poly-1 and poly-2, followed by assaying them against wild-type and drug-resistant influenza A Wuhan viruses. Methods: Individually or together, 1 and 2 were covalently bonded to the polymer. The antiviral potencies of the resultant poly-1, poly-2, poly-(1 + 2), and poly-1 + poly-2, as well as 1 and 2, were assessed using plaque reduction assay. Results: Attaching 1 to the polymer improved at best millimolar IC50 values over three orders of magnitude. While 2 exhibited micromolar IC50 values, poly-2 was >100-fold even more potent. The IC50 of poly-(1 + 2) against the wild-type strain was >300-fold and ∼17-fold better than of poly-1 and poly-2, respectively. In contrast, the potency of poly-(1 + 2) vs. poly-2 against the mutant strain merely doubled. The mixture of poly-1 + poly-2 inhibited both viral strains similarly to poly-2. Conclusions: The bifunctional poly-(1 + 2) acts synergistically against the wild-type influenza virus, but not against its drug-resistant mutant, as compared to a physical mixture of the monofunctional poly-1 and poly-2., National Institutes of Health (U.S.) (NIH grant U01-AI074443), Fundación Ramón Areces (postdoctoral fellowship)