Structure, rheology, and copper-complexation of a hyaluronan-like exopolysaccharide from Vibrio

MO245 exopolysaccharide (EPS) was produced in laboratory conditions from Vibrio genus microorganism isolated from bacterial mats found in Moorea Island. Its structure consists of a linear tetrasaccharide repeating unit →4)-β-D-GlcpA-(1→4)-α-D-GalpNAc-(1→3)-β-D-GlcpNAc-(1→4)-β-D-GlcpA-(1→ containing covalently-linked 5% of glucose, galactose, and rhamnose, determined by methylation analyses and NMR spectroscopy. The molecular weight, radius of gyration (Rg) and intrinsic viscosity, [η], determined by gel permeation chromatography with light scattering and viscosity detection, were 513 ± 4 kDa (PDI, 1.42 ± 0.01), 6.7 ± 0.3 dl/g and 56 ± 0.3 nm respectively. The chelation of the EPS with copper divalent ions leads to the instantaneous formation of gels. The structural similitude proposed, based in an equal ratio of GlcA to N-acetylated sugars and in the same type of glyosidic linkages present in the repeating unit (alternated 1→3 and 1→4 linkages), is translated into analogous physicochemical properties: MO245 EPS is a flexible polyelectrolyte, with scaling exponents similar to that described for HA. This similitude opens opportunities in future drug delivery, tissue engineering, and cosmetic applications.publishe

CARB 113: Co-assembly of peptide and carbohydrate amphiphiles to generate proteoglycan mimics

Peptide amphiphiles (PA) have been used as building blocks that generate nanofibrous protein mimics through self-assembly under physiological conditions. These supramolecular structures are maintained by non-covalent interactions, such as, Pi-Pi stacking, hydrogen bonding and hydrophobic effects. The generated fibers can be further crosslinked via salt bridges thus forming hydrated systems that resemble the extracellular matrix (ECM) at structural and functional level. However, the proteins in the ECM are often presented as glycoconjugates such as glycoproteins and proteoglycans. Carbohydrate-modified PAs are just emerging as alternative or complementary building blocks able to generate closer supramolecular ECM mimics. Such PAs are challenging at synthetic, supramolecular and biofunctional level. Carbohydrates bear different â OH groups prompt to react and thus, different protections are needed for selective functionalization. Moreover, once conjugated to the PA, the carbohydrate moiety can alter its self-assembling capacity, as well as, the biofunctionality of the incorporated bioactive peptide. We therefore developed a simpler approach for generation of minimalistic proteoglycan mimics: co-assembly of short, aromatic PA and their carbohydrate analogues. The nanofibers generated by this approach have a PA core (e.g. fmoc-FF) and a carbohydrate shell (e.g. fmoc-glucosamine-6-phosphate or fmoc-glucosamine-6-sulfate). They present: 1) a higher mechanical performance than the PA single component systems; 2) an improved biofunctionality as demonstrated by our studies with growth factors (e.g. FGF2), lectins and cells. Peptide amphiphiles (PA) have been used as building blocks that generate nanofibrous protein mimics through self-assembly under physiological conditions. These supramolecular structures are maintained by non-covalent interactions, such as, Pi-Pi stacking, hydrogen bonding and hydrophobic effects. The generated fibers can be further crosslinked via salt bridges thus forming hydrated systems that resemble the extracellular matrix (ECM) at structural and functional level. However, the proteins in the ECM are often presented as glycoconjugates such as glycoproteins and proteoglycans. Carbohydrate-modified PAs are just emerging as alternative or complementary building blocks able to generate closer supramolecular ECM mimics. Such PAs are challenging at synthetic, supramolecular and biofunctional level. Carbohydrates bear different –OH groups prompt to react and thus, different protections are needed for selective functionalization. Moreover, once conjugated to the PA, the carbohydrate moiety can alter its self-assembling capacity, as well as, the biofunctionality of the incorporated bioactive peptide. We therefore developed a simpler approach for generation of minimalistic proteoglycan mimics: co-assembly of short, aromatic PA and their carbohydrate analogues. The nanofibers generated by this approach have a PA core (e.g. fmoc-FF) and a carbohydrate shell (e.g. fmoc-glucosamine-6-phosphate or fmoc-glucosamine-6-sulfate). They present: 1) a higher mechanical performance than the PA single component systems; 2) an improved biofunctionality as demonstrated by our studies with growth factors (e.g. FGF2), lectins and cells.  info:eu-repo/semantics/publishedVersio

Minimalistic supramolecular proteoglycan mimics by co-assembly of aromatic peptide and carbohydrate amphiphiles

We report the co-assembly of aromatic carbohydrate and dipeptide amphiphiles under physiological conditions as a strategy to generate minimalistic proteoglycan mimics. The resulting nanofibers present a structural, fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) core and a functional carbohydrate (Fmoc-glucosamine-6-sulfate or -phosphate) shell. The size, degree of bundling and mechanical properties of the assembled structures depend on the chemical nature of the carbohydrate amphiphile used. In cell culture medium, these nanofibers can further organize into supramolecular hydrogels. We demonstrate that, similar to proteoglycans, the assembled gels prolong the stability of growth factors and preserve the viability of cultured cells. Our results demonstrate that this approach can be applied to the design of extracellular matrix (ECM) substitutes for future regenerative therapies.We acknowledge the EU's H2020 and FP7 framework programmes (Forecast 668983; CHEM2NATURE 692333; THE DISCOVERIES CTR 739572; ERC AdG ComplexiTE 321266) and the Portuguese FCT (IF/00032/2013; BD/113794/2015; BPD/85790/2012; M-ERA-NET2/0001/2016 – INCIPIT; ENMed/001/2015 – CytoNanoHeal). We thank P. Frederix for his help in the FTIR measurements and M. Mullin for her help in the TEM imaging.info:eu-repo/semantics/publishedVersio

End-on PEGylation of heparin: Effect on anticoagulant activity and complexation with protamine

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijbiomac.2023.125957.Heparin is the most common anticoagulant used in clinical practice but shows some downsides such as short half- life (for the high molecular weight heparin) and secondary effects. On the other hand, its low molecular weight analogue cannot be neutralized with protamine, and therefore cannot be used in some treatments. To address these issues, we conjugated polyethylene glycol (PEG) to heparin reducing end (end-on) via oxime ligation and studied the interactions of the conjugate (Hep-b-PEG) with antithrombin III (AT) and protamine. Isothermal titration calorimetry showed that Hep-b-PEG maintains the affinity to AT. Dynamic light scattering demonstrated that the Hep-b-PEG formed colloidal stable nanocomplexes with protamine instead of large multi-molecular aggregates, associated with heparin side effects. The in vitro (human plasma) and in vivo experiments (Sprague Dawley rats) evidenced an extended half-life and higher anticoagulant activity of the conjugate when compared to unmodified heparin.  The authors thank INNO Laboratório Veterinário for the measurements of in vivo coagulation times, Raul Pacheco for discussions about the ITC results, Teresa Oliveira for her help in the in vivo experiments and Ramón Rail for his help in the 3D structures of Fig. 1, Fig. 4. We thank funding provided by the Portuguese Foundation for Science and Technology (PTDC/QUI-POL/28117/2017 and CEECIND/00814/2017). África González-Fernández thanks Xunta de Galicia (Grupo de Referencia competitiva, GRC-ED431C 2020/02) 2020-2023

Preparation, properties and bioapplications of block copolymers nanopatterns

Block copolymer (BCP) self-assembly has emerged as a feasible method for large-scale fabrication with remarkable precision â features that are not common for most of the nanofabrication techniques. In this review, we present recent advancements in the molecular design of BCP along with state-of-the-art processing methodologies based on microphase separation alone or its combination with different lithography methods. Furthermore, we explore the bioapplications of the generated nanopatterns in the development of protein arrays, cell-selective surfaces, and antibacterial coatings. Finally, we outline the current challenges in the field and discuss the potential breakthroughs that can be achieved by adopting BCP approaches already applied in the fabrication electronic devices.We acknowledge the support from the Portuguese Foundation for Science and Technology (grants PTDC/QUI-POL/28117/2017, PD/BD/128085/2016, and CEECIND/00814/2017) and the European fund for regional development via the Operational Programme for Competitiveness and Internalization (grant POCI-01-0145-FEDER-028117)

Disclosing an NMR-Invisible Fraction in Chitosan and PEGylated Copolymers and Its Role on the Determination of Degrees of Substitution

An unexpected ¹H NMR invisible fraction (IF) for chitosan (CS) and CS<i>-g-</i>PEG is reported. The presence of this IF is remarkable considering that solution NMR is recognized as the method of choice for studying structural modifications in CS, including the degrees of acetylation (DA) and substitution (DS). In spite of IF figures as high as 50%, this IF does not interfere in the correct determination of the DA by ¹H NMR, pointing to a homogeneous distribution of acetyl groups along the visible and invisible fractions. Quite in contrast, the IF negatively biases the determination of the DS in CS<i>-g-</i>PEG, with relative errors as high as 150% in a broad range of temperatures, pH values, and concentrations. This fact raises concerns about the accuracy of previously reported DS data for CS<i>-g-</i>PEG and many other CS copolymers. Efficient user-friendly conditions have been developed for the correct determination of the DS of CS<i>-g-</i>PEG by depolymerization by nitrous acid

Antithrombotic and hemocompatible properties of nanostructured coatings assembled from block copolymers

We describe the antithrombotic properties of nanopatterned coatings created by self-assembly of poly(styrene-block-2-vinylpyridine) (PS-b-P2VP) with different molecular weights. By changing the assembly conditions, we obtained nanopatterns that differ by their morphology (size and shape of the nanopattern) and chemistry. The surface exposition of P2VP block allowed quaternization, i.e. introduction of positive surface charge and following electrostatic deposition of heparin. Proteins (albumin and fibrinogen) adsorption, platelet adhesion and activation, cytocompatibility, and reendothelization capacity of the coatings were assessed and discussed in a function of the nanopattern morphology and chemistry. We found that quaternization results in excellent antithrombotic and hemocompatible properties comparable to heparinization by hampering the fibrinogen adhesion and platelet activation. In the case of quaternization, this effect depends on the size of the polymer blocks, while all heparinized patterns had similar performance showing that heparin surface coverage of 40 % is enough to improve substantially the hemocompatibility.Portuguese Foundation for Science and Technology for the grants PTDC/QUI-POL/28117/2017, PD/BD/128085/2016, and CEECIND/00814 /2017

Block copolymer nanopatterns affect cell spreading: stem versus cancer bone cells

Bone healing after a tumor removal can be promoted by biomaterials that enhance the bone regeneration and prevent the tumor relapse. Herein, we obtained several nanopatterns by self-assembly of polystyrene-block-poly 2-vinyl pyridine (PS-b-P2VP) with different molecular weight and investigated the adhesion and morphology of human bone marrow mesenchymal stem cells (BMMSC) and osteosarcoma cell line (SaOS-2) on these patterns aiming to identify topography and chemistry that promote bone healing. We analyzed > 2000 cells per experimental condition using imaging software and different morphometric descriptors, namely area, perimeter, aspect ratio, circularity, surface/area, and fractal dimension of cellular contour (FDC). The obtained data were used as inputs for principal component analysis, which showed distinct response of BMMSC and SaOS-2 to the surface topography and chemistry. Among the studied substrates, micellar nanopatterns assembled from the copolymer with high molecular weight promote the adhesion and spreading of BMMSC and have an opposite effect on SaOS-2. This nanopattern is thus beneficial for bone regeneration after injury or pathology, e.g. bone fracture or tumor removal.Portuguese Foundation for Science and Technology for the grants PTDC/QUI-POL/28117/2017, PD/BD/128085/2016, and CEECIND/00814 /201