Anti-Metastatic Properties of a Marine Bacterial Exopolysaccharide-Based Derivative Designed to Mimic Glycosaminoglycans

Osteosarcoma is the most frequent malignant primary bone tumor characterized by a high potency to form lung metastases. In this study, the effect of three oversulfated low molecular weight marine bacterial exopolysaccharides (OS-EPS) with different molecular weights (4, 8 and 15 kDa) were first evaluated in vitro on human and murine osteosarcoma cell lines. Different biological activities were studied: cell proliferation, cell adhesion and migration, matrix metalloproteinase expression. This in vitro study showed that only the OS-EPS 15 kDa derivative could inhibit the invasiveness of osteosarcoma cells with an inhibition rate close to 90%. Moreover, this derivative was potent to inhibit both migration and invasiveness of osteosarcoma cell lines; had no significant effect on their cell cycle; and increased slightly the expression of MMP-9, and more highly the expression of its physiological specific tissue inhibitor TIMP-1. Then, the in vivo experiments showed that the OS-EPS 15 kDa derivative had no effect on the primary osteosarcoma tumor induced by osteosarcoma cell lines but was very efficient to inhibit the establishment of lung metastases in vivo. These results can help to better understand the mechanisms of GAGs and GAG-like derivatives in the biology of the tumor cells and their interactions with the bone environment to develop new therapeutic strategies

Sterilization of Exopolysaccharides Produced by Deep-Sea Bacteria: Impact on Their Stability and Degradation

Polysaccharides are highly heat-sensitive macromolecules, so high temperature treatments are greatly destructive and cause considerable damage, such as a great decrease in both viscosity and molecular weight of the polymer. The technical feasibility of the production of exopolysaccharides by deep-sea bacteria Vibrio diabolicus and Alteromonas infernus was previously demonstrated using a bioproduct manufacturing process. The objective of this study was to determine which sterilization method, other than heat sterilization, was the most appropriate for these marine exopolysaccharides and was in accordance with bioprocess engineering requirements. Chemical sterilization using low-temperature ethylene oxide and a mixture of ionized gases (plasmas) was compared to the sterilization methods using gamma and beta radiations. The changes to both the physical and chemical properties of the sterilized exopolysaccharides were analyzed. The use of ethylene oxide can be recommended for the sterilization of polysaccharides as a weak effect on both rheological and structural properties was observed. This low-temperature gas sterilizing process is very efficient, giving a good Sterility Assurance Level (SAL), and is also well suited to large-scale compound manufacturing in the pharmaceutical industry

The Potential Biotechnological Applications of the Exopolysaccharide Produced by the Halophilic Bacterium Halomonas almeriensis

We have studied the extracellular polysaccharide (EPS) produced by the type strain, M8T, of the halophilic bacterium Halomonas almeriensis, to ascertain whether it might have any biotechnological applications. All the cultural parameters tested influenced both bacterial growth and polysaccharide production. EPS production was mainly growth-associated and under optimum environmental and nutritional conditions M8T excreted about 1.7 g of EPS per litre of culture medium (about 0.4 g of EPS per gram of dry cell weight). Analysis by anion-exchange chromatography and high-performance size-exclusion chromatography indicated that the exopolysaccharide was composed of two fractions, one of 6.3 × 106 and another of 1.5 × 104 Daltons. The monosaccharide composition of the high-molecular-weight fraction was mannose (72% w/w), glucose (27.5% w/w) and rhamnose (0.5% w/w). The low-molecular-weight fraction contained mannose (70% w/w) and glucose (30% w/w). The EPS has a substantial protein fraction (1.1% w/w) and was capable of emulsifying several hydrophobic substrates, a capacity presumably related to its protein content. The EPS produced solutions of low viscosity with pseudoplastic behaviour. It also had a high capacity for binding some cations. It contained considerable quantities of sulphates (1.4% w/w), an unusual feature in bacterial polysaccharides. All these characteristics render it potentially useful as a biological agent, bio-detoxifier and emulsifier.This research was supported by grants from the Dirección General de Investigación Científica y Técnica BOS 2003-00498; CGL2005-05947; CGL2008-02399; BIO2011-2013 and by grants from the Plan Andaluz de Investigación PO6-CVI-01850

Anticoagulant potential of modified sulfated exopolysaccharides from deep-sea bacteria: toward non-animal heparin alternatives

Heparin, a widely used polysaccharidic anticoagulant of animal origin, is associated with risks of contamination and adverse effects, notably bleeding and thrombocytopenia. These limitations have prompted interest in alternative sulfated polysaccharides with anticoagulant properties and improved safety profiles. This study explored the anticoagulant potential of two marine bacterial exopolysaccharides (EPS), infernan and diabolican. It assessed whether chemical modifications (depolymerization, oversulfation) could enhance their anticoagulant properties compared to unfractionated and low molecular weight heparins. Native EPS were depolymerized to generate different molecular weights and then chemically oversulfated to increase negative charge density. Anticoagulant activities were evaluated using clotting and thrombin generation assays (TGA). Molecular docking was performed to model interactions with antithrombin and heparin cofactor II. Only highly sulfated derivatives significantly prolonged activated partial thromboplastin time while showing negligible effect on thrombin time and anti-factor Xa activity. They present different structures, and their binding to antithrombin is not achieved via the classic pentasaccharide motif. In TGA, these derivatives inhibited thrombin formation at higher doses than heparin but induced a marked delay in clot generation. Docking analyses supported their ability to bind serpins, albeit with lower specificity than heparin. Their limited anti-Xa activity and non-animal origin position them as promising anticoagulant candidates

Antiviral Activities of Sulfated Polysaccharides Isolated from Sphaerococcus coronopifolius (Rhodophytha, Gigartinales) and Boergeseniella thuyoides (Rhodophyta, Ceramiales)

Water-soluble sulfated polysaccharides isolated from two red algae Sphaerococcus coronopifolius (Gigartinales, Sphaerococcaceae) and Boergeseniella thuyoides (Ceramiales, Rhodomelaceae) collected on the coast of Morocco inhibited in vitro replication of the Human Immunodeficiency Virus (HIV) at 12.5 μg/mL. In addition, polysaccharides were capable of inhibiting the in vitro replication of Herpes simplex virus type 1 (HSV-1) on Vero cells values of EC50 of 4.1 and 17.2 μg/mL, respectively. The adsorption step of HSV-1 to the host cell seems to be the specific target for polysaccharide action. While for HIV-1, these results suggest a direct inhibitory effect on HIV-1 replication by controlling the appearance of the new generations of virus and potential virucidal effect. The polysaccharides from S. coronopifolius (PSC) and B. thuyoides (PBT) were composed of galactose, 3,6-anhydrogalactose, uronics acids, sulfate in ratios of 33.1, 11.0, 7.7 and 24.0% (w/w) and 25.4, 16.0, 3.2, 7.6% (w/w), respectively

Marine Polysaccharides in Pharmaceutical Applications: An Overview

The enormous variety of polysaccharides that can be extracted from marine plants and animal organisms or produced by marine bacteria means that the field of marine polysaccharides is constantly evolving. Recent advances in biological techniques allow high levels of polysaccharides of interest to be produced in vitro. Biotechnology is a powerful tool to obtain polysaccharides from a variety of micro-organisms, by controlling the growth conditions in a bioreactor while tailoring the production of biologically active compounds. Following an overview of the current knowledge on marine polysaccharides, with special attention to potential pharmaceutical applications and to more recent progress on the discovering of new polysaccharides with biological appealing characteristics, this review will focus on possible strategies for chemical or physical modification aimed to tailor the final properties of interest

Les sucres de l'extrême pour la médecine de demain

À la fin des années 1980, l'Ifremer a initié des recherches sur les micro-organismes des écosystèmes hydrothermaux océaniques profonds. Parmi les échantillons prélevés – environ 1330 isolats –, des bactéries productrices de biopolymères d’intérêt biotechnologique, comme des exopolysaccharides (EPS) et des polyhydroxyalcanaotes (PHA), ont été identifiées. De genres connus, elles appartiennent à des espèces ignorées jusqu’alors (Alteromonas infernus, Alteromonas macleodii subsp. fijiensis biovar deepsane ou Vibrio diabolicus)

Préparation, caractérisation et activités biologiques de fucanes extraits d'algues brunes

Fucoldans are a family of high molecular weight sulfated polysaccharides (from 8x105 to 1 x106 g/mol) widely dispersed in brown seaweed cell wall. When extracted from several brown algae, they exhibit anticoagulant properties. The chemical degradation of a crude extract, from Pelvetia canaliculata undertaken to obtain a low molecular weight polysaccharide (4 x 104 g/mol) with the purpose of a possible clinical use. Its anticoagulant potency was investigated through the inhibition of factor IIa and factor Xa in the presence of antithrombin III or heparin cofactor II. The degraded fucoidan revealed a potent antithrombin activity, although weaker than heparin. In the presence of either antithrombin III depleted plasma or purified heparin cofactor II, the fucoidan was as efficient as heparin or dermatan sulfate on heparin cofactor II potentiation, at the same ponderal concentration. In whole plasma or in presence of the purified inhibitor, an inhibition of factor Ila activity mediated by antithrombin III was detected (30 times less potent than heparin, on a weight to weight basis). No inhibition of factor Xa activity was detected in the same experimental conditions, Chemical degradations of various crude extracts, from four brown seaweeds; have given low molecular weight fucoidan fractions (almost 2 x 104 g/mol). In plasma or in presence of purified heparin cofactor II, these fractions have revealed a same anticoagulant activity and mode of action that the previous fraction extracted from Pelvetia canaliculata. These fucoidans, by-products of alginates preparation in the food and cosmetologic industries, are obtained easily. Thus, they may represent a cheap and easy source of a new type of anticoagulants.Les fucanes, polysaccharides sulfatés extraits de la paroi extracellulaire des algues brunes sont décrits depuis plus d'une trentaine d'années comme présentant une activité anticoagulante vis à vis du plasma sanguin. Le mécanisme d'action du fucane sur le système de la coagulation sanguine n'a pas donné lieu à des études très approfondies jusqu'en 1989. Après l'extraction et la caractérisation de fucanes issus de différentes algues brunes, les polysaccharides de très haute masse molaire (1 x 105 g/mol) isolés ont montré des propriétés anticoagulantes sur plasma pauvre en plaquettes qui confirment les travaux antérieurs. Une étude approfondie du mécanisme de l'activité anticoagulante a été ensuite réalisée sur une fraction de fucane dégradée par hydrolyse acide et bien caractérisée de masse molaire de 40 000 g/mol. Cette étude a montré que le fucane de faible masse molaire dénommé F2 accélère l'inhibition de la thrombine, principale enzyme procoagulante du système de la coagulation, par l'intermédiaire de ses deux principaux inhibiteurs physiologiques qui sont l'antithrombine III et le deuxième cofacteur de l'héparine. Aucune accélération de l'inhibition du facteur Xa, autre enzyme procoagulante de la coagulation, n'a été détectée en plasma pauvre en plaquettes. L'originalité du fucane étudié réside dans le fait qu'il inhibe fortement la thrombine en présence du deuxième cofacteur de l'héparine et pour des concentrations pondérales équivalentes à celles de l'héparine. L'autre originalité du fucane est due aussi à sa capacité, à même concentration pondérale, d'inactiver spécifiquement la thrombine par l'intermédiaire du deuxième cofacteur de l'héparine et de l'antithrombine III. La méthode de dégradation par hydrolyse acide comparée à une autre méthode de dégradation par radiolyse a montré que ces deux méthodes conduisaient à des fucanes de faible masse molaire conservant en grande partie l'activité anticoagulante initiale. La préparation d'un fucane "optimal" par dégradation acide, à partir de l'algue Ascophyllum nodosum, a permis l'obtention d'une fraction de fucane dénommée D2, de masse molaire de 18 000 g/mol. L'étude in vivo réalisée chez le lapin montre que cette fraction est plus active, par rapport à l'héparine, en expérimentation in vivo qu'en essais in vitro

Skin tissue engineering using functional marine biomaterials

International audienceThe development of new skin substitutes is based on the construction of a scaffold (such as films, hydrogels, plates, sheets, fibers, sponges). Numerous hydrogels are already used and the polymers, which are able to form hydrogels, are either from a natural origin (hyaluronic acid, alginate, chitosan, chondroitin sulfate, collagen, etc.) or from a synthetic one (polylactide-co-glycolic, polyethylene glycol, etc.). The therapeutic potential of natural bioactive compounds such as polysaccharides (especially glycosaminoglycans or GAGs) is now well documented, and this activity combined with their natural biodiversity will allow the development of a new generation of therapeutics. Marine environment can offer a large variety of GAG-like molecules from various origins (animals, seaweeds, invertebrates, microorganisms, etc.), which could be a good alternative to the use of mammalian GAGs as bioactive components for tissue regeneration. Recent studies have been made on newly described marine bacteria isolated from hydrothermal deep-sea vents and able to produce extracellular polysaccharides (EPS) with unusual structures. These bacterial EPS and their derivatives offer great advantages over both mammalian GAGs and other traditional marine polysaccharides. These biotechnologically produced marine GAG-like molecules and their derivatives present often very original structures and have a better benefit/risk ratio than GAGs from mammalian origins (e.g., heparin)