Nicotine is a potent extracellular polysaccharide inducer in Fusobacterium nucleatum biofilms

The purpose of this in vitro study was to analyze the influence of nicotine on the extracellular polysaccharides in Fusobacterium nucleatum biofilm. Methods: F. nucleatum (ATCC 10953) biofilms supplemented with different concentrations of nicotine (0, 0.5, 1, 2, 4, and 8 mg/mL) were grown in two different BHI broth conditions [no sucrose and 1% sucrose]. Extracellular polysaccharides assay, pH measurements, and a spectrophotometric assay were performed. Data were submitted for ANOVA and Tukey honestly significant difference analyses (HSD) tests (α =.05). Results: Extracellular polysaccharides synthesis was influenced by an interaction between nicotine concentrations and growth medium solution containing sucrose (P<.05). The pH values declined in the sucrose-exposed biofilm were greater than in the group exposed only to nicotine (P<.05). The biofilm exposed to sucrose and nicotine had a higher total biofilm growth (P<.05) than the nicotine-treated biofilm without sucrose. Conclusions: Regardless of sucrose exposure, biofilms exposed to different nicotine concentrations influenced the amount of extracellular polysaccharides

Freeze-thaw and solvent-exchange strategy to generate physically cross-linked organogels and hydrogels of curdlan with tunable mechanical properties.

Physical gels from natural polysaccharides present the advantage of no toxic cross-linking agents and no chemical modification during preparation. Herein, novel physical gels, transparent organogels and opaque hydrogels from the microorganism-derived (1,3)-ß-D-glucan of curdlan were prepared in dimethyl sulfoxide (DMSO) using the freeze-thaw technique, followed by a solvent-exchange strategy with water. The mechanical and structural properties of these gels were investigated by rheology, scanning electron microscopy, attenuated total reflection infrared spectroscopy, wide-angle X-ray diffraction and small-angle X-ray scattering. Gelation mechanisms and intermolecular interaction models have also been proposed. The good solvent DMSO serves as both a crosslinker and a pore-foaming agent in organogels. The reversible macromolecular conformation changes and phase separation of curdlan endow the gels with reversible transparency, volume change and tunable mechanical strength. The new design strategy of facile preparation and performance tuning provides a platform for developing new organogels and sterile hydrogels of curdlan.

Developing an Effective Glycan-Based Vaccine for Streptococcus Pyogenes.

Streptococcus pyogenes is a primary infective agent that causes approximately 700 million human infections each year, resulting in more than 500 000 deaths. Carbohydrate-based vaccines are proven to be one of the most promising subunit vaccine candidates, as the bacterial glycan pattern(s) are different from mammalian cells and show increased pathogen serotype conservancy than the protein components. In this Review we highlight reverse vaccinology for use in the development of subunit vaccines against S. pyogenes, and report reproducible methods of carbohydrate antigen production, in addition to the structure-immunogenicity correlation between group A carbohydrate epitopes and alternative vaccine antigen carrier systems. We also report recent advances used to overcome hurdles in carbohydrate-based vaccine development.

Physicochemical and biological characterization of a xanthan gum-polyvinylpyrrolidone hydrogel obtained by gamma irradiation.

Xanthan gum (XG) and polyvinylpyrrolidone (PVP) are two polymers with low toxicity, high biocompatibility, biodegradability, and high hydrophilicity, making them promising candidates for multiple medical aspects. The present work aimed to synthesize a hydrogel from a mixture of XG and PVP and crosslinked by gamma irradiation. We assessed the hydrogel through a series of physicochemical (FT-IR, TGA, SEM, and percentage of swelling) and biological (stability of the hydrogel in cell culture medium) methods that allowed to determine its applicability. The structural evaluation by infrared spectrum demonstrated that a crosslinked hydrogel was obtained from the combination of polymers. The calorimetric test and swelling percentage confirmed the formation of the bonds responsible for the crosslinked structure. The calorimetric test evidenced that the hydrogel was resistant to decomposition in contrast to non- irradiated material. The determination of the swelling degree showed constant behavior over time, indicating a structure resistant to hydrolysis. This phenomenon also occurred during the test of stability in a cell culture medium. Additionally, microscopic analysis of the sample revealed an amorphous matrix with the presence of porosity. Thus, the findings reveal the synthesis of a novel material that has desirable attributes for its potential application in pharmaceutical and biomedical areas.

Preparation and evaluation of gellan gum hydrogel reinforced with silk fibers with enhanced mechanical and biological properties for cartilage tissue engineering.

Various research about cartilage regeneration using biomaterials has been done recently. Particularly, gellan gum hydrogel (GG) is reported to be suitable as a biomaterial for cartilage tissue engineering (TE) for its water uptaking ability, producibility, and environmental resemblance of native cartilage. Despite these advantages, mechanical and cell adhesion properties are still difficult to modulate. Reinforcement is essential to overcome these problems. Herein, GG was modified by physically blending with different lengths of silk fiber (SF). As SF is expected to improve such disadvantages of GG, mechanical and biological properties were characterized to confirm its reinforcement ability. Mechanical properties such as degradation rate, swelling rate, compression strength, and viscosity were studied and it was confirmed that SF significantly reinforces the mechanical properties of GG. Furthermore, in vitro study was carried out to confirm morphology, biocompatibility, proliferation, and chondrogenesis of chondrocytes encapsulated in the hydrogels. Overall, chondrocytes in the GG blended with SF (SF/GG) showed enhanced cell viability and growth. According to this study, SF/GG can be a promising biomaterial for cartilage TE biomaterial.

Chemical modification strategies for viscosity-dependent processing of gellan gum.

Recently, the hydrogel-forming polysaccharide gellan gum (GG) has gained popularity as a versatile biomaterial for tissue engineering purposes. Here, we examine the modification strategies suitable for GG to overcome processing-related limitations. We emphasize the thorough assessment of the viscoelastic and mechanical properties of both precursor solutions and final hydrogels. The investigated modification strategies include purification, oxidation, reductive chain scission, and blending. We correlate polymer flow and hydrogel forming capabilities to viscosity-dependent methods including casting, injection and printing. Native GG and purified NaGG are shear thinning and feasible for printing, being similar in gelation and compression behavior. Oxidized GGox possesses reduced viscosity, higher toughness, and aldehydes as functional groups, while scissored GGsciss has markedly lower molecular weight. To exemplify extrudability, select modification products are printed using an extrusion-based bioprinter utilizing a crosslinker bath. Our robust modification strategies have widened the processing capabilities of GG without affecting its ability to form hydrogels.

Formulation development and in-vitro evaluation of gastroretentive drug delivery system of loxoprofen sodium: A natural excipients based approach.

The limitations of conventional type delivery systems to retain drug (s) in the stomach has resulted in the development of novel gastroretentive drug delivery system. We developed single-layer effervescent floating tablets of loxoprofen sodium for prolong delivery in the stomach using natural polymers xanthan gum, guar gum and semisynthetic polymer HPMCK4M. All the formulations (F1-F9) were developed by varying concentrations of xanthan gum and HPMCK4M while guar gum concentration was kept constant. Two gas generating agent (s) incorporated were sodium bicarbonate and citric acid. All compendial pre and post-compression tests results were in the acceptable limits. FTIR analysis confirmed drug-polymer compatibility. The in-vitro drug release in simulated conditions i.e., 0.1 N HCl for 12 h revealed orderly increase in total floating time, i.e., less than 6 h for F1 over 12 h for F9. Formulations F1 to F4 were not capable to retard drug release up to 12 h, whereas F5-F7 for 12 h, while F8 and F9 for more than 12 h. Data fitting in various kinetic models showed that drug release best fit in first order kinetic model and F9 in zero order. Based on results data, F7 was the best among all.

Bacterial surface capsular polysaccharides from Streptococcus pneumoniae: A systematic review on structures, syntheses, and glycoconjugate vaccines.

The polysaccharide capsule of Streptococcus pneumoniae constitutes the outermost surface structure of the organism and plays a critical role in virulence. The capsule is the target of current pneumococcal vaccines and glycoconjugates and has important medical and industrial applications. Widespread use of these vaccines is driving changes in serotype prevalence in disease. A massive array of sugars and glycosidic linkages experienced with complete diversity of potential polysaccharide structures. However, it is impossible to collect a sufficient quantity of glycan antigens for the preparation of CPS-based glycoconjugate vaccines from natural sources with high purity and for thorough biological evaluation. So nowadays, the development of a chemical synthetic strategy and their conjugation with a carrier protein to form synthetic glycoconjugate vaccines has been used to gain access on a large scale. This review provides a comprehensive summary of structures, synthesis as well as recent development of synthetic glycoconjugate vaccines, which will support research and may benefit the glycochemical and medical sciences.

Preparation and characterisation of a gellan gum-based hydrogel enabling osteogenesis and inhibiting Enterococcus faecalis.

Infections are the leading cause of failure of osteogenic material implantation. Antibiotic treatment, treatment with bone cement, or collagen sponge placement can result in drug resistance and difficulties in operation. To address this, gellan gum (GG) was selected in this study and prepared as an injectable hydrogel containing chlorhexidine (CHX) and nanohydroxyapatite (nHA) that overcomes these intractable problems. Scanning electron microscopy and micro-computed tomography revealed a three-dimensional polymeric network of the hydrogel. The hydrogel had excellent biocompatibility, as detected by cell counting kit-8 and Live/Dead assay. Bone marrow mesenchymal stem cells could be encapsulated into the network, showing that the structure was suitable for cell growth. Additionally, loading the hydrogel with nHA improved its mechanical, biodegradable, and osteogenic properties. Quantitative alkaline phosphatase and Alizarin Red S staining validated its osteogenic ability. Furthermore, antibacterial activity assessment showed that the hydrogel loaded with 50 µg/mL CHX inhibited Enterococcus faecalis in a concentration-dependent manner. Thus, we report an injectable GG-based hydrogel with superior antibacterial effect against E. faecalis and osteogenesis, which holds promise for treating infectious bone defects caused by refractory periradicular periodontitis.

A stabilized glycomimetic conjugate vaccine inducing protective antibodies against Neisseria meningitidis serogroup A.

Neisseria meningitidis serogroup A capsular polysaccharide (MenA CPS) consists of (1 → 6)-2-acetamido-2-deoxy-α-D-mannopyranosyl phosphate repeating units, O-acetylated at position C3 or C4. Glycomimetics appear attractive to overcome the CPS intrinsic lability in physiological media, due to cleavage of the phosphodiester bridge, and to develop a stable vaccine with longer shelf life in liquid formulation. Here, we generate a series of non-acetylated carbaMenA oligomers which are proven more stable than the CPS. An octamer (DP8) inhibits the binding of a MenA specific bactericidal mAb and polyclonal serum to the CPS, and is selected for further in vivo testing. However, its CRM197 conjugate raises murine antibodies towards the non-acetylated CPS backbone, but not the natural acetylated form. Accordingly, random O-acetylation of the DP8 is performed, resulting in a structure (Ac-carbaMenA) showing improved inhibition of anti-MenA CPS antibody binding and, after conjugation to CRM197, eliciting anti-MenA protective murine antibodies, comparably to the vaccine benchmark.

Silver nanocomposites based on the bacterial fucose-rich polysaccharide secreted by Enterobacter A47 for wound dressing applications: Synthesis, characterization and in vitro bioactivity.

This study demonstrates the potential of a high molecular weight fucose-containing polysaccharide secreted by the bacterium Enterobacter A47, named FucoPol, and its silver nanocomposite as potential bioactive materials for wound dressings applications. A green, simple, light-assisted method was used for the synthesis of silver nanoparticles (AgNP) using FucoPol, as stabilizing and reducing agent. The synthesized nanoparticles were spherical, and the main population had a particle size in number ranging between 13 and 30 nm for percentiles 50 and 90, respectively. FucoPol, as well as the functionalized material, besides having no cytotoxicity towards human skin keratinocytes and mouse fibroblasts, also promoted in vitro keratinocytes migration. These observations not only show the safety of FucoPol and FucoPol/AgNP biocomposite, but also their wound healing ability. Moreover, the biocomposite had a strong antimicrobial activity against Staphylococcus aureus ATCC 6538 and Klebsiella pneumoniae CECT 8453, two representative strains of known skin commensal pathogens. These findings demonstrate for the first time the potential of FucoPol for the development of wound healing formulations. Additionally, the FucoPol/AgNP biocomposite might find use in antimicrobial biomaterials, including antibacterial wound healing formulations, which further strengthens the establishment of FucoPol as a bioactive biopolymer.

Synthesis and biological evaluation of a bioinspired, tissue-adhesive gellan gum-based hydrogel designed for minimally invasive delivery and retention of chondrogenic cells.

A dopamine-modified, bioinspired gellan gum hydrogel (STM-148B) with improved physicochemical and biological characteristics, suitable for minimally invasive cell delivery and retention in the context of cartilage repair, is herein presented. STM-148B's putative game-changing design characteristics include a highly biocompatible, animal-free and chemically defined composition, reproducibility of manufacture and ease of formulation. STM-148B undergoes rapid ionic crossinking by physiologically relevant mono and divalent cations to form stable 3D hydrogels that possess excellent tissue adhesiveness, such that additional fixation aids are rendered superfluous. STM-148B hydrogels maintain viability of mammalian cells and further promote up-regulation of the expression of healthy chondrogenic extracellular matrix markers upon stimulation. STM-148B is currently undergoing pre-clinical safety and efficacy assessment as a medical device for cell delivery and retention focussing on regeneration of hyaline-like cartilage and may represent a valuable addition to the armamentarium of tissue-engineering therapies for treatment of focal cartilage lesions.

Efficient Strategy for α-Selective Glycosidation of d-Glucosamine and Its Application to the Synthesis of a Bacterial Capsular Polysaccharide Repeating Unit Containing Multiple α-Linked GlcNAc Residues.

An efficient α-selective glycosylation method was developed for the synthesis of 2-deoxy-2-amino-d-glucosides based on synergetic α-directing effects of the TolSCl/AgOTf promotion system and the functional groups at the corresponding azido donor 6-O-position to exert steric ß-shielding effect or remote participation in the glycosylation reaction. Its practicability was verified with a wide range of monosaccharide glycosyl acceptors and the first, one-pot synthesis of the challenging pentasaccharide repeating unit of an Acinetobacter baumannii K47 capsular polysaccharide.

Staphylococcus aureus capsular polysaccharides: a structural and synthetic perspective.

The S. aureus bacterium is surrounded by capsular polysaccharides. These capsular polysaccharides are important in the pathogenesis of staphylococcal infection. There are 11 serotypes of capsular polysaccharides that have been identified, and a majority of strains express capsular polysaccharides type 5 (CP5) or 8 (CP8). The main focus of this review is to describe recent advances in the area of the chemical synthesis of monosaccharide components of S. aureus CP, oligosaccharide assembly and functionalization. Chemical routes to obtain oligosaccharides derived from CP1, CP5 and CP8 represent a compendium of modern classics of the total synthesis of challenging glycan sequences.

Broadly protective semi-synthetic glycoconjugate vaccine against pathogens capable of producing poly-ß-(1→6)-N-acetyl-d-glucosamine exopolysaccharide.

Poly-ß-(1→6)-N-acetylglucosamine (PNAG) was first discovered as a major component of biofilms formed by Staphylococcus aureus and some other staphylococci but later this exopolysaccharide was also found to be produced by pathogens of various nature. This common antigen is considered as a promising target for construction of a broadly protective vaccine. Extensive studies of PNAG, its de-N-acetylated derivative (dPNAG, containing around 15% of residual N-acetates) and their conjugates with Tetanus Toxoid (TT) revealed the crucial role of de-N-acetylated glucosamine units for the induction of protective immunity. Conjugates of synthetic penta- (5GlcNH2) and nona-ß-(1→6)-d-glucosamines (9GlcNH2) were tested in vitro and in different animal models and proved to be effective in passive and active protection against different microbial pathogens. Presently conjugate 5GlcNH2-TT is being produced under GMP conditions and undergoes safety and effectiveness evaluation in humans and economically important animals. Current review summarizes all stages of this long-termed study.

Enhancing biocompatibility and neuronal anti-inflammatory activity of polymyxin B through conjugation with gellan gum.

Polymyxins, as strong antibiotics with high liposaccharide deactivation abilities, are rarely used as neuronal anti-inflammatory agent because of their high cytotoxicity. In this study, polymyxin B (PMB) was conjugated with deacylated gellan gum for the sustained release of PMB to reduce its cytotoxicity at high concentration without affecting the antibacterial and liposaccharide binding activities. For the conjugate of original PMB/GN ratio of 1.0 (GPC), the conjugating rate was 96.40%, and the releasing ratio of PMB was 30.12% within 60 h. The FT-IR spectra of GPC indicated that the amino groups of PMB were covalently bonded with the COOH groups of gellan and other PMB molecules. Most GPCs were micelle shaped regardless of whether they were under dry conditions or in an aqueous solution. The inhibition zones of PMB against Escherichia coli and Pseudomonas aeruginosa were small, but the half maximal inhibitory concentration value against BV-2 cells increased from 15.63 µg/mL to 2000.00 µg/mL after conjugation. GPC can also effectively depress the liposaccharide-stimulated overexpression of cytotoxic nitric oxide by BV-2 cells. This study revealed the possibility of using polymyxins for neuronal anti-inflammation and that this gellan/PMB conjugate can potentially be applied to wound healing and implants.

Structural features of microbial exopolysaccharides in relation to their antioxidant activity.

Oxidative damage caused by free radicals is an inevitable and pervasive phenomenon that leads to cell damage and the emergence of diseases including ageing, cancer, diabetes, cardiovascular disease and neurodegenerative disorders. In this context, antioxidants play a significant role in encountering free radicals by delaying or reducing the oxidative damage of cells. Evidence suggests that synthetic antioxidants are double-edged swords wherefore the requirement for natural antioxidants is increasing globally. Exploring non-toxic, biodegradable and compatible natural molecules like exopolysaccharides can favour the current antioxidant limitations. Microbial exopolysaccharides represent a structurally diverse class of carbohydrate molecules secreted at the cell wall. Recently, bioprospecting exopolysaccharides for their astounding physiochemical properties and the reliable structure-activity relationship have motivated more research towards the investigation of their antioxidant properties. Here we propose that structural features of exopolysaccharides such as monosaccharide residues, branching, molecular weight, glycosidic linkage, functional groups, protein, selenium, and chemical modifications are likely to influence their antioxidant activity. To support this hypothesis we review the interdependence of structural features of exopolysaccharides to the observed antioxidant activity. In light of its importance, this review focuses on the understanding of the elimination of free radicals by microbial exopolysaccharides derived from marine and nonmarine sources during the last six years.

Regioselective Glycosylation Strategies for the Synthesis of Group Ia and Ib Streptococcus Related Glycans Enable Elucidating Unique Conformations of the Capsular Polysaccharides.

Group B Streptococcus serotypes Ia and Ib capsular polysaccharides are key targets for vaccine development. In spite of their immunospecifity these polysaccharides share high structural similarity. Both are composed of the same monosaccharide residues and differ only in the connection of the Neu5Acα2-3Gal side chain to the GlcNAc unit, which is a ß1-4 linkage in serotype Ia and a ß1-3 linkage in serotype Ib. The development of efficient regioselective routes for GlcNAcß1-3[Glcß1-4]Gal synthons is described, which give access to different group B Streptococcus (GBS) Ia and Ib repeating unit frameshifts. These glycans were used to probe the conformation and molecular dynamics of the two polysaccharides, highlighting the different presentation of the protruding Neu5Acα2-3Gal moieties on the polysaccharide backbones and a higher flexibility of Ib polymer relative to Ia, which can impact epitope exposure.

Selectively Charged and Zwitterionic Analogues of the Smallest Immunogenic Structure of Streptococcus Pneumoniae Type 14.

Zwitterionic polysaccharides (ZPs) have been shown in recent years to display peculiar immunological properties, thus attracting the interest of the carbohydrate research community. To fully elucidate the mechanisms underlying these properties and exploit the potential of this kind of structures, in depth studies are still required. In this context, the preparation of two cationic, an anionic, as well as two zwitterionic tetrasaccharide analogues of the smallest immunogenic structure of Streptococcus pneumoniae type 14 (SP14) capsular polysaccharide are presented. By exploiting a block strategy, the negative charge has been installed on the non-reducing end of the lactose unit of the tetrasaccharide and the positive charge either on the non-reducing end of the lactosamine moiety or on an external linker. These structures have then been tested by competitive ELISA, showing that the structural variations we made do not modify the affinity of the neutral compound to binding to a specific antibody. However, lower efficacies than the natural SP14 compound were observed. The results obtained, although promising, point to the need to further elongate the polysaccharide structure, which is likely too short to cover the entire epitopes.

Levofloxacin Hemihydrate In Situ Gelling Ophthalmic Solution: Formulation Optimization and In Vitro and In Vivo Evaluation.

Bacterial conjunctivitis is a leading cause of ocular infections requiring short-term therapeutic treatment with frequent administration of drugs on daily basis. Topical dosage forms available in the market for the treatment of bacterial conjunctivitis such as simple drug solutions and suspensions are rapidly eliminated from the precorneal space upon instillation due to tear turn over and nasolacrimal drainage, limiting intraocular bioavailability of drug to less than 10% of the administered dose. To overcome issues related to conventional drop, an effort was made to design and evaluate prolong release ophthalmic solution of levofloxacin hemihydrate (LFH) using ion-sensitive in situ gelling polymer. Gellan gum was used as the in situ gelling agent. Formulations were screened based on in vitro gelation time, in vitro drug release, and stability towards sol to gel conversion upon storage. The prototype formulations exhibiting quick in vitro gelling time (< 15 s), prolonged in vitro drug release (18-24 h), and stability for at least 6 months at 25°C/40% relative humidity (RH) and 40°C/25% RH were evaluated for pharmacokinetic studies using healthy New Zealand white rabbits. Tested formulations were found to be well-tolerated and showed significant increase in AUC0-24 (22,660.39 h ng/mL) and mean residence time (MRT 12 h) as compared with commercially available solution Levotop PF® (Ajanta Pharma Ltd., India)(AUC0-24 6414.63 h ng/mL and MRT 4 h). Thus, solution formulations containing in situ gelling polymer may serve as improved drug delivery system providing superior therapeutic efficacy and better patient compliance for the treatment of bacterial conjunctivitis.