Anti-inflammatory and antibacterial activities of cerium-containing mesoporous bioactive glass nanoparticles for drug-free biomedical applications.

Mesoporous bioactive glass nanoparticles (MBGNPs) are attracting significant attention as suitable materials for multifunctional biomedical applications. In this study, cerium was incorporated into MBGNPs using two different approaches. In the first approach, cerium was added to the glass system directly during the synthesis, while in the second approach, cerium was added to the as-synthesized MBGNPs via the template ion-exchange method. The influence of the method of synthesis on the physicochemical properties of nanoparticles was examined by SEM, TEM, XRD, FTIR, and N2 adsorption-desorption analyses. The MBGNPs exhibited spheroidal morphology and disordered mesoporous structure. XRD analysis confirmed the amorphous nature of the nanoparticles. The chemical composition was determined by the acid digestion method using ICP-OES. The influence of the synthesis method on the specific surface area, mesoporosity, and solubility of synthesized nanoparticles in Tris/HCl (pH 7.4) and acetate (pH 4.5) buffer has also been studied. The obtained Ce containing MBGNPs were non-cytotoxic toward â€‹preosteoblast MC3T3-E1 cells in contact with nanoparticles in a concentration of up to 100 â€‹µg/mL. The anti-inflammatory effect of Ce containing MBGNPs was tested with lipopolysaccharides (LPS)-induced proinflammatory RAW 264.7 macrophage cells. Ce containing MBGNPs decreased the release of nitric oxide, indicating the anti-inflammatory response of macrophage cells. Ce containing MBGNPs also showed antibacterial activity against S. aureus and E. coli. The mentioned features of the obtained MBGNPs make them useful in a variety of biomedical applications, considering their biocompatibility, anti-inflammatory response, and enhanced antibacterial effect.

14-3-3ε protein-immobilized PCL-HA electrospun scaffolds with enhanced osteogenicity.

Adipose-derived mesenchymal stem cells (ASCs) accelerate the osteointegration of bone grafts and improve the efficiency in the formation of uniform bone tissue, providing a practical and clinically attractive approach in bone tissue regeneration. In this work, the effect of nanofibrous biomimetic matrices composed of poly(ε-caprolactone) (PCL), nanometric hydroxyapatite (nHA) particles and 14-3-3 protein isoform epsilon on the initial stages of human ASCs (hASCs) osteogenic differentiation was investigated. The cells were characterized by flow cytometry and induction to differentiation to adipogenic and osteogenic lineages. The isolated hASCs were induced to differentiate to osteoblasts over all scaffolds, and adhesion and viability of the hASCs were found to be similar. However, the activity of alkaline phosphatase (ALP) as early osteogenic marker in the PCL-nHA/protein scaffold was four times higher than in PCL-nHA and more than five times than the measured in neat PCL.

Development of a bioactive glass-polymer composite for wound healing applications.

This study reports the production and characterization of a composite material for wound healing applications. A bioactive glass obtained by sol-gel process and doped with two different metal ions was investigated. Silver (Ag) and cobalt (Co) were chosen due to their antibacterial and angiogenic properties, respectively, very beneficial in the wound healing process. Poly(ε-caprolactone) (PCL) fibers were produced by electrospinning (ES) from a polymeric solution using acetone as a solvent. After optimization of the ES parameters, two main suspensions were prepared, namely: PCL containing bioactive glass nanoparticles (BG-NP) and PCL with Ag2O and CoO doped BG-NP (DP BG-NP), which were processed with different concentrations of BG-NP (0.25%, 0.5% and 0.75wt%). The composite membranes were characterized in terms of morphology, fiber diameter, weight loss, mineralization potential and mechanical performance.

Fractions of chemically oversulphated galactosaminoglycan sulphates inhibit three enveloped viruses: human immunodeficiency virus type 1, herpes simplex virus type 1 and human cytomegalovirus.

A series of chemically oversulphated galactosaminoglycans (SO3H:COOH ratio > or = 2) were tested in vitro as antiviral agents against human immunodeficiency virus type 1 (HIV-1), the aetiological agent of AIDS, and against herpes simplex virus type 1 and human cytomegalovirus, two agents responsible for opportunistic infections in HIV-infected people. The oversulphated derivatives displayed an increase in activity ranging from one to four orders of magnitude against the three viruses, as compared to the natural parent compounds (SO3H:COOH, ratio approx. 1). The antiviral activity of these polyanions appears to be favoured by a high degree of sulphation and a high molecular mass. An oversulphated dermatan, with a SO3H:COOH ratio of 2.86 and molecular mass of 23.2 kDa, was the most potent anti-HIV-1 compound (EC50 0.04 microgram/ml). A second oversulphated dermatan, with a SO3H:COOH ratio of 2.40 and molecular mass of 25 kDa, displayed the highest activity against HSV-1 (EC50 0.01 microgram/ml). An oversulphated chondroitin, with a SO3H:COOH ratio of 2.80 and molecular mass of 17.3 kDa, was the strongest anti-HCMV agent (EC50 0.4 microgram/ml). In view of the absence of the side-effects typical of heparin-like compounds, a combination of these derivatives could have therapeutic potential.

Heterogeneity of unfractionated heparins studied in connection with species, source, and production processes.

The heterogeneity of unfractionated heparins (Hep) can be correlated to the species and organs of origin and to the process of production. Heparins, extracted by different, validated processes from different organs and/or tissues (mucosa, thymus, pancreas, placenta, lung, intestine) or mammals (pig, beef, sheep, man) and other vertebrates (chicken), have been examined by HPLC analysis of heparinase digests. By analysis of disaccharides many observations have been made. Porcine mucosa heparin (pm-Hep) was always found to contain higher amounts of the disaccharides delta UA-GlcNS,6S and delta UA-2S-GlcNS,6S, than did bovine mucosa heparin (bm-Hep), whereas bm-Hep always showed higher amounts of the sequence IdoA(2OSO3)-GlcNSO3 than did pm-Hep. These findings mean that the last step of the biosynthesis, the 6-O-sulfation of glucosamine-N-sulfate (GlcNSO3), is accomplished; in bm-Hep, to a lesser extent than in pm-Hep. The 6-O-sulfated molar fractions of pig mucosa, chicken intestine, beef pancreas, beef placenta, and beef lung heparins were higher than the corresponding molar fractions of beef mucosa and beef thymus Heps. Also the manufacturing processes can partially rearrange the heparin structure. Even 6-O-sulfation enrichment (by chromatographic purification) or base-catalyzed displacement of sulfate groups from IdoA2SO3 occurred. The resulting anticoagulant activity roughly correlated with the percentage of trisulfated disaccharide and the 6-O-sulfated molar fraction. The heparin from human placenta was similar to pm-Hep. The observed species- and organ-dependent structural characteristics support the suggestion by Nader and Dietrich (in Heparin, Chemical and Biological Properties, Lane DA, U Lindahl (Eds). Arnold, London, 1989, p 81) on the antipathogenic role of heparin. The 6-O-sulfation of glucosamine, present in higher amounts in organs that function as barriers against many foreign bodies, like lung, placenta, intestine of chicken and pig, may play an important role in this antipathogenic action of Hep.

Active site for heparin cofactor II in low molecular mass dermatan sulfate. Contribution to the antithrombotic activity of fractions with high affinity for heparin cofactor II.

Dermatan sulfate (DS) is currently under clinical investigation as new antithrombotic agent. Unlike heparin, DS does not act through Antithrombin III (ATIII) but primarily through thrombin on Heparin Cofactor II (HCII). HCII is activated by the oversulfated sequence (IdoA2SO3-GalNAc4SO3)4 or by both the sequences (IdoA2SO3-GalNAc4SO3)n and (IdoA-GalNAc-4,6SO3)n, [n > or = 2]. A Low Molecular Mass Dermatan Sulfate (LMM-DS), endowed with a bioavailability three-four times higher than DS, by subcutaneous route, was obtained by chemical depolymerization of DS. The LMM-DS was fractionated by anion exchange and size exclusion chromatography. Fractions with high and low charge densities, high and low molecular masses, and high (2.66) and low (0.07) potencies on HCII were isolated. A relationship between the in vitro HCII-mediated inhibition of thrombin and the chain length of DS fractions containing oversulfated sequences was found [by a multiple regression test]. The in vivo activity increased until it reached a plateau. The important influence on the HCII activity of natural IdoA-GalNAc-4,6SO3 disaccharide was confirmed by investigation on oversulfated DS obtained by a limited and selective chemical 6-O-sulfation in GalNAc4SO3 units of DS.

Analysis of heparin origin by HPLC quantitation of disaccharide components.

A simple method was developed to analyze by high performance liquid chromatography unsaturated disaccharide isomers, derived from heparin by enzymatic digestion. This method was successfully exploited in the investigation of heparin origin. The percent amount of 6-sulphated disaccharides in the heparin extracted from porcine mucosa was found to be higher than that contained in the heparin extracted from bovine mucosa. The differences in the contents of the disaccharides obtained by enzymatic beta-elimination cleavage of heparin were confirmed by 13C-NMR measures of heparin in toto. The processes for extracting and purifying heparin may, however, modify the sulphation pattern of heparin. The structure of the latter seems to depend on the species owing to the specificity of the biosynthesis.

Inhibition of human leukocyte elastase by chemically and naturally oversulfated galactosaminoglycans.

Several samples of oversulfated chondroitin and dermatan were obtained by chemical sulfation and by SAX-HPLC enrichment. The starting products and oversulfated products were tested as potential inhibitors of human leukocyte elastase, an enzyme hypothesized to be involved in the etiology of diseases such as emphysema, atherosclerosis, and rheumatoid arthritis. Chemical oversulfation (SO3H/COOH 1.6-3.2), preferentially occurring at C-6 of galactosamine residues, was found generally to increase the inhibitory power on elastase. Chemically oversulfated galactosaminoglycans thus have potential as therapeutic agents, considering that they produce non-significant effects on the hemocoagulative system. Two naturally oversulfated dermatans sulfate (SO3H/COOH ca. 1.2), mainly oversulfated at C-2 of iduronic acid residues, showed comparatively higher anticoagulant activity (in the HC-II mediated thrombin inhibition test).

Quantitation of dermatan sulfate active site for heparin cofactor II by 1H nuclear magnetic resonance spectroscopy.

The sequence (IdoA2SO3-GalNAc4SO3)n contributes to the HCII-mediated inhibition of thrombin by dermatan sulfate (DS). This sequence clearly results from the 13C NMR spectrum and can be quantified by the signal C1-H of IdoA2SO3 in the 1H NMR spectrum. A linear correlation has been found between the content in the disulfated disaccharide delta Di-2,4diS obtained by enzymatic demolition with ABC lyase, the percentage content in IdoA2SO3 quantified by 1H NMR, and the HCII-mediated activity of dermatan sulfates from beef mucosa and pig skin. DSs have been obtained also from pig mucosa and contain an amount, not negligible, of delta Di-4, 6diS. This disulfate disaccharide contributes to the activity expressed by the IdoA2SO3-GalNAc4SO3 sequence. The analytical techniques HPLC and 1H NMR, applied to the currently performed analyses of DS, are described and discussed.

Relative influence of different disulphate disaccharide clusters on the HCII-mediated inhibition of thrombin by dermatan sulphates of different origins.

Besides the major monosulphated disaccharide sequences (IdoA-GalNAc4SO3), dermatan sulphates (DS) contain the oversulphated sequences (IdoA2SO3-GalNAc4SO3) and (IdoA-GalNAc4, 6SO3), the concentration of which is correlated with the HCII-mediated inhibition of thrombin by DS. The effect of the chemical removal of the sulphate groups on the HCII-mediated activity was studied. The base-catalyzed sulphate group displacement from IdoA2SO3 residues, leading to formation of the epoxyde aGulA, is a 1st order reaction. When the content of sequences (IdoA-GalNAc4, 6SO3) is higher than that of sequences (IdoA2SO3-GalNAc4SO3), removal of the sulphate groups from Ido2SO3 reduces the HCII activity less than when the latter sequences prevail. (IdoA-GalNAc4, 6SO3) cooperates with (IdoA2SO3-GalNAc4SO3) in the activation of the HCII. Also the removal of 6-SO3- groups from GalNAc4, 6SO3, in absence of IdoA2SO3-GalNAc4SO3, considerably reduces the activity. A low molecular mass natural fraction rich in IdoA2SO3 as well as glucuronic acid, having higher electrophoretic mobility than the higher molecular mass DS which contains less glucuronic acid, is remarkably active.

Structure and contribution to the heparin cofactor II-mediated inhibition of thrombin of naturally oversulphated sequences of dermatan sulphate.

Dermatan sulphate (DS) obtained from bovine and pig mucosa and pig skin, and charge-enriched fractions of a selected DS preparation, were characterized in terms of charge density, M(r) and disaccharide composition of chondroitin ABC lyase digests, and by 13C-n.m.r. spectroscopy. Besides the major IdoA-GalNAc4SO3 sequences, all DS preparations contain about 10% disulphated disaccharide sequences (mostly IdoA2SO3-GalNAc4SO3, with minor amounts of IdoA-GalNAc4,6SO3). DS fragments (prepared by radical-catalysed depolymerization of DS and retaining the internal structure of the parent polysaccharide) as well as Smith degraded fragments [SD-DS, obtained by controlled degradation of periodate-oxidized and borohydride-reduced DS (RO-DS)] with the general structure GalNAc4SO3(IdoA2SO3-GalNAc4SO3)n-R (where R is the remnant of a glycol-split uronic acid, and n = 2-3 and 3-4) were characterized by one- and two-dimensional 1H-n.m.r., 13C-n.m.r. and disaccharide composition analysis. In accordance with previous findings [Maimone and Tollefsen (1990) J. Biol. Chem. 265, 18263-18271], only fragments with n > or = 3 significantly enhance the heparin cofactor II-mediated inhibition of thrombin. In natural DS preparations and their fractions, this activity (as well as the antithrombotic activity in an animal model) appears to require IdoA2SO3-containing sequences. The heparin cofactor II activity of DS, RO-DS and SD-DS fragments decreases with decreasing M(r). However, RO-DS fragments are more active than DS fragments of similar M(r), probably because of the extra flexibility endowed by glycol-split IdoA residues.