Monitoring the stability of heparin: NMR evidence for the rearrangement of sulfated iduronate in phosphate buffer.

Heparin, a major anticoagulant drug, comprises a complex mixture of motifs. Heparin is isolated from natural sources while being subjected to a variety of conditions but the detailed effects of these on heparin structure have not been studied in depth. Therefore, the result of exposing heparin to a range of buffered environments, ranging pH values from 7 to 12, and temperatures of 40, 60 and 80 °C were examined. There was no evidence of significant N-desulfation or 6-O-desulfation in glucosamine residues, nor of chain scission, however, stereochemical re-arrangement of α-L-iduronate 2-O-sulfate to α-L-galacturonate residues occurred in 0.1 M phosphate buffer at pH 12/80 °C. The results confirm the relative stability of heparin in environments like those during extraction and purification processes; on the other hand, the sensitivity of heparin to pH 12 in buffered solution at high temperature is highlighted, providing an important insight for heparin manufacturers.

Influence of cations, pH and dispersed phases on pectin emulsification properties.

The cooperativity of six cations (Ca2+, Mg2+, Zn2+, Al3+, Cr3+ and Fe3+), three pectins (sugar beet, high and low methyl esterified), three dispersed phases (medium chain triglycerides (MCT), orange oil and hexadecane), time (30 days) and pH (2.0 and 6.0) has been investigated in the formation and stability against coarsening of oil-in-water emulsions. Cations generally influenced emulsion stability in the following order (most stable) Ca2+ â€‹> â€‹Mg2+ â€‹> â€‹Al3+ â€‹> â€‹Cr3+ â€‹> â€‹Zn2+ â€‹> â€‹Fe3+ (least stable). This order largely coincided with that of the strength of pectin-cation interactions showing that the higher the affinity of cation for pectin the less stable the emulsion. More stable emulsions were formed with sugar beet pectin, which was also unresponsive to the presence of cations, followed by high- and then low-methyl esterified samples. At pH 2.0 all pectins showed their best emulsification performance whereas shifting pH to 6.0 severely impaired emulsification capacity and longer term stability against droplet growth. Smaller droplets were created with hexadecane under all conditions studied followed by MCT and orange oil in agreement with their aqueous solubilities. The present results advance our understanding of the stabilisation of emulsions using pectin and allow us to tailor their functionality for applications in food, pharmaceutical and biomedical industries.

Hydrolytic Degradation of Heparin in Acidic Environments: Nuclear Magnetic Resonance Reveals Details of Selective Desulfation.

Heparin is a complex glycosaminoglycan, derived mainly from pig mucosa, used therapeutically for its anticoagulant activity. Yet, owing largely to the chain complexity, the progressive effects of environmental conditions on heparin structure have not been fully described. A systematic study of the influence of acidic hydrolysis on heparin chain length and substitution has therefore been conducted. Changes in the sulfation pattern, monitored via 2D NMR, revealed initial de-N-sulfation of the molecule (pH 1/ 40 °C) and unexpectedly identified the secondary sulfate of iduronate as more labile than the 6-O-sulfate of glucosamine residues under these conditions (pH 1/ 60 °C). Additionally, the loss of sulfate groups, rather than depolymerization, accounted for most of the reduction in molecular weight. This provides an alternative route to producing partially 2-O-de-sulfated heparin derivatives that avoids using conventional basic conditions and may be of value in the optimization of processes associated with the production of heparin pharmaceuticals.

The identification and characterisation of novel bioactive peptides derived from porcine liver.

Bioactive peptides (BAPs) can be derived from a variety of sources; these could be from dietary proteins which are then broken down in the gastrointestinal tract to release BAPs, or they can be isolated from various sources ex vivo. Sources include plant-based proteins such as soy, and chickpeas, and animal proteins from waste from the meat industry and from fish skin. Bioinformatics is also a useful approach to assess the peptides released from digests due to the great number of possible sequences that can be isolated from proteins. Therefore, an in silico analysis of peptides could potentially lead to a more rapid discovery of BAPs. This article investigates a "crude" liver peptide mixture derived from papain hydrolysis of porcine liver and purified peptides derived from the hydrolysates following HPLC fractionation and in silico digestion of the host proteins identified using LC-MS/MS. This allowed the identification of two proteins (cytosol aminopeptidase and haemoglobin subunit alpha) present in the "crude" mixture after LC-MS/MS. In silico hydrolysis of these proteins identified that several peptides were predicted to be both present in the crude mixture using the BIOPEP database and to have potential bioactivity using the Peptide Ranker tool. Peptides (FWG, MFLG and SDPPLVFVG) with the greatest potential bioactivity and which had not previously been reported in the literature were then synthesised. The results indicated that the predicted bioactivity of the synthetic peptides would likely include antioxidant activity. FWG and MFLG derived from the in silico papain hydrolysis of cytosol aminopeptidase showed activity better or comparable to Trolox in the Oxygen Radical Absorbance Capacity (ORAC) assay. The use of these in silico tools, alongside a robust range of biochemical assays which cover a wider range of bioactivities would be a way of improving the discovery of novel bioactive peptides.

Fluorescent Dye Labeling Changes the Biodistribution of Tumor-Targeted Nanoparticles.

Fluorescent dye labeling is a common strategy to analyze the fate of administered nanoparticles in living organisms. However, to which extent the labeling processes can alter the original nanoparticle biodistribution has been so far neglected. In this work, two widely used fluorescent dye molecules, namely, ATTO488 (ATTO) and Sulfo-Cy5 (S-Cy5), have been covalently attached to a well-characterized CXCR4-targeted self-assembling protein nanoparticle (known as T22-GFP-H6). The biodistribution of labeled T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5 nanoparticles has been then compared to that of the non-labeled nanoparticle in different CXCR4+ tumor mouse models. We observed that while parental T22-GFP-H6 nanoparticles accumulated mostly and specifically in CXCR4+ tumor cells, labeled T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5 nanoparticles showed a dramatic change in the biodistribution pattern, accumulating in non-target organs such as liver or kidney while reducing tumor targeting capacity. Therefore, the use of such labeling molecules should be avoided in target and non-target tissue uptake studies during the design and development of targeted nanoscale drug delivery systems, since their effect over the fate of the nanomaterial can lead to considerable miss-interpretations of the actual nanoparticle biodistribution.

An Auristatin nanoconjugate targeting CXCR4+ leukemic cells blocks acute myeloid leukemia dissemination.

BACKGROUND: Current acute myeloid leukemia (AML) therapy fails to eliminate quiescent leukemic blasts in the bone marrow, leading to about 50% of patient relapse by increasing AML burden in the bone marrow, blood, and extramedullar sites. We developed a protein-based nanoparticle conjugated to the potent antimitotic agent Auristatin E that selectively targets AML blasts because of their CXCR4 receptor overexpression (CXCR4+) as compared to normal cells. The therapeutic rationale is based on the involvement of CXCR4 overexpression in leukemic blast homing and quiescence in the bone marrow, and the association of these leukemic stem cells with minimal residual disease, dissemination, chemotherapy resistance, and lower patient survival. METHODS: Monomethyl Auristatin E (MMAE) was conjugated with the CXCR4 targeted protein nanoparticle T22-GFP-H6 produced in E. coli. Nanoconjugate internalization and in vitro cell viability assays were performed in CXCR4+ AML cell lines to analyze the specific antineoplastic activity through the CXCR4 receptor. In addition, a disseminated AML animal model was used to evaluate the anticancer effect of T22-GFP-H6-Auristatin in immunosuppressed NSG mice (n = 10/group). U of Mann-Whitney test was used to consider if differences were significant between groups. RESULTS: T22-GFP-H6-Auristatin was capable to internalize and exert antineoplastic effects through the CXCR4 receptor in THP-1 and SKM-1 CXCR4+ AML cell lines. In addition, repeated administration of the T22-GFP-H6-Auristatin nanoconjugate (9 doses daily) achieves a potent antineoplastic activity by internalizing specifically in the leukemic cells (luminescent THP-1) to selectively eliminate them. This leads to reduced involvement of leukemic cells in the bone marrow, peripheral blood, liver, and spleen, while avoiding toxicity in normal tissues in a luminescent disseminated AML mouse model. CONCLUSIONS: A novel nanoconjugate for targeted drug delivery of Auristatin reduces significantly the acute myeloid leukemic cell burden in the bone marrow and blood and blocks its dissemination to extramedullar organs in a CXCR4+ AML model. This selective drug delivery approach validates CXCR4+ AML cells as a target for clinical therapy, not only promising to improve the control of leukemic dissemination but also dramatically reducing the severe toxicity of classical AML therapy.

Caffeine release and absorption from caffeinated gums.

The objectives of this study were to estimate the impact of chewing time on caffeine release from gum and to understand caffeine pharmacokinetics. Caffeine release increased with chewing time (2 min < 5 min < 10 min). Furthermore, two plasma caffeine concentration peaks were observed suggesting that caffeine absorption occurs both through the oral mucosa and gastrointestinal tract. This is of practical relevance to maximise caffeine doses and to synchronise effort with peak caffeine concentration.

Evaluation of the mucoadhesive properties of chitosan nanoparticles prepared using different chitosan to tripolyphosphate (CS:TPP) ratios.

Mucoadhesive molecules such as chitosan, can allow targeting of a particular tissue to prolong residence time and subsequently improve bioavailability. The purpose of this study was to investigate chitosan-tripolyphosphate (CS:TPP) nanoparticles and to evaluate the interaction between nanoparticles of different CS:TPP ratios with mucin using viscosity, particle size analysis and ζ-potential. For all CS:TPP ratios examined, a minimum value of viscosity was reached for a 3:1 CS:TPP ratio, however chitosan nanoparticles at this ratio were not stable (<+30 mV), whereas a CS:TPP ratio of 4:1 displayed the strongest interaction. This suggests a minimum CS:TPP ratio of 4:1 is required to produce stable nanoparticles able to form strong interactions, which is consistent with a greater mucin binding efficiencies at CS:TPP ratios of 4:1 and higher, which were quantified using a colorimetric assay. Further analysis of similar systems could lead potentially to tuneable chitosan nanoparticles for specific applications.

Structural and rheological studies of a polysaccharide mucilage from lacebark leaves (Hoheria populnea A. Cunn.).

A water-soluble mucilage extracted from the leaves of Hoheria populnea was chemically and physically studied. Monosaccharide composition and linkages were determined by high performance anion exchange chromatography (HPAEC), gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Lacebark mucilage was composed of rhamnose, galactose, galacturonic acid and glucuronic acid (2:1:2:1). Proton and 13C NMR spectroscopy, and linkage analysis, revealed a predominantly rhamnogalacturonan I-type (RG I-type) structure comprising of a backbone of →4]-α-D-GalpA-[1→2]-α-L-Rhap-[1→. Data indicated the mucilage likely comprises of a polymer containing several structurally discrete domains or possibly more than one discrete polymer. One domain contains a RG I-type backbone with branching at O-3 of GalpA residues to terminal ß-D-GlcpA residues, another similarly contains a RG I-type backbone but is branched at O-4 of the Rhap residues to terminal GalpA residues or oligosaccharides containing α-linked 4-Galp and terminal GalpA residues. A possible third domain contains contiguous 2-Rhap residues, some branched at O-3. Hydrated mucilage exhibited pseudoplastic flow behaviour and viscoelastic properties of an entangled biopolymer network. These rheological behaviours were only slightly affected by pH and may prove advantageous in potential end-product applications including oral pharmaceuticals or as a food ingredient.

Comparative Study of Diethylaminoethyl-Chitosan and Methylglycol-Chitosan as Potential Non-Viral Vectors for Gene Therapy.

In this paper, we compared the transfection efficiency and cytotoxicity of methylglycol-chitosan (MG-CS) and diethylaminoethyl-chitosan (DEAE-CSI and DEAE-CSII with degrees of substitution of 1.2 and 0.57, respectively) to that of Lipofectamine (used as a reference transfection vector). MG-CS contains quaternary amines to improve DNA binding, whereas the DEAE-CS exhibits pH buffering capability that would ostensibly enhance transfection efficiency by promoting endosomal escape. Gel retardation assays showed that both DEAE-CS and MG-CS bound to DNA at a polysaccharide:DNA mass ratio of 2:1. In Calu-3 cells, the DNA transfection activity was significantly better with MG-CS than with DEAE-CS, and the efficiency improved with increasing polysaccharide:DNA ratios. By contrast, the efficiency of DEAE-CSI and DEAE-CSII was independent of the polysaccharide:DNA ratio. Conversely, in the transfection-recalcitrant JAWSII cells, both Lipofectamine and MG-CS showed significantly lower DNA transfection activity than in Calu-3 cells, whereas the efficiency of DEAE-CSI and DEAE-CSII was similar in both cell lines. The toxicity of DEAE-CS increased with increasing concentrations of the polymer and its degree of substitution, whereas MG-CS demonstrated negligible cytotoxicity, even at the highest concentration studied. Overall, MG-CS proved to be a more efficient and less toxic transfection agent when compared to DEAE-CS.

Designing chitosan-tripolyphosphate microparticles with desired size for specific pharmaceutical or forensic applications.

Chitosan (CS) is a natural cationic polymer obtained by the partial N-deacetylation of chitin. Chitosan microparticles can be prepared by cross-linking with tripolyphosphate (TPP) via the ionic interaction between positively charged amino groups (CS) and negatively charged counter ions (TPP). This can be controlled by the charge density of CS and TPP, which depend on the pH and ionic strength of the solution. The purpose of this study is to investigate the combined effects of three independent variables (pH, ionic strength and CS:TPP ratio) on three important physico-chemical properties (viscosity, zeta potential and particle size) during the preparation of microparticles. CS:TPP microparticles were prepared using experimental design and equations were generated and used to predict relative viscosity, zeta potential and particle size under different conditions. This gives us the ability to design tuneable CS-TPP microparticles with desired size for specific pharmaceutical or forensic applications e.g. latent fingerprint visualisation.

Solution conformation and flexibility of capsular polysaccharides from Neisseria meningitidis and glycoconjugates with the tetanus toxoid protein.

The structural integrity of meningococcal native, micro-fluidized and activated capsular polysaccharides and their glycoconjugates - in the form most relevant to their potential use as vaccines (dilute solution) - have been investigated with respect to their homogeneity, conformation and flexibility. Sedimentation velocity analysis showed that the polysaccharide size distributions were generally bimodal with some evidence for higher molar mass forms at higher concentration. Weight average molar masses Mw where lower for activated polysaccharides. Conjugation with tetanus toxoid protein however greatly increased the molar mass and polydispersity of the final conjugates. Glycoconjugates had an approximately unimodal log-normal but broad and large molar mass profiles, confirmed by sedimentation equilibrium "SEDFIT MSTAR" analysis. Conformation analysis using HYDFIT (which globally combines sedimentation and viscosity data), "Conformation Zoning" and Wales-van Holde approaches showed a high degree of flexibility - at least as great as the unconjugated polysaccharides, and very different from the tetanus toxoid (TT) protein used for the conjugation. As with the recently published finding for Hib-TT complexes, it is the carbohydrate component that dictates the solution behaviour of these glycoconjugates, although the lower intrinsic viscosities suggest some degree of compaction of the carbohydrate chains around the protein.

The physicochemical characterisation of pepsin degraded pig gastric mucin.

Mucins are the main macromolecular components of the mucus secretions that cover the oral cavity, gastrointestinal and urogenital tracts of animals. The properties of the mucus secretions are therefore directly correlated with the physicochemical properties of mucin glycoproteins. In this study, mucins were obtained from pig gastric mucous after digestion with pepsin at 37°C for 4h, these mucins were characterised in terms of compositional and hydrodynamic properties. Compositional analysis showed that this mucin contains protein (15%), carbohydrates (55%) of which the constituents are: fucose (4%), galactose (9%), glucosamine (55%), glucosamine (33%) and sialic acid (2%). The latter component gives the mucin polymer a pH-dependant negative charge, with a ζ-potential of -3mV at pH 1.2 up to -11mV at pH 7.4. The weight average molar mass was ∼1×10(6)g/mol and intrinsic viscosity was ∼0.42dL/g although there was a small pH dependency due to the polyelectrolyte behavior of the polymer. The measurements of viscosity versus shear rate showed shear thinning behavior and the critical overlap concentration was determined to be 10-11% w/v indicating a compact structure. Knowledge of these properties is fundamental to the understanding interactions of mucins, with for example, novel drug delivery systems.

A glycoconjugate of Haemophilus influenzae Type b capsular polysaccharide with tetanus toxoid protein: hydrodynamic properties mainly influenced by the carbohydrate.

Three important physical properties which may affect the performance of glycoconjugate vaccines against serious disease are molar mass (molecular weight), heterogeneity (polydispersity), and conformational flexibility in solution. The dilute solution behaviour of native and activated capsular polyribosylribitol (PRP) polysaccharides extracted from Haemophilus influenzae type b (Hib), and the corresponding glycoconjugate made by conjugating this with the tetanus toxoid (TT) protein have been characterized and compared using a combination of sedimentation equilibrium and sedimentation velocity in the analytical ultracentrifuge with viscometry. The weight average molar mass of the activated material was considerably reduced (Mw ~ 0.24 × 10(6) g.mol(-1)) compared to the native (Mw ~ 1.2 × 10(6) g.mol(-1)). Conjugation with the TT protein yielded large polydisperse structures (of Mw ~ 7.4 × 10(6) g.mol(-1)), but which retained the high degree of flexibility of the native and activated polysaccharide, with frictional ratio, intrinsic viscosity, sedimentation conformation zoning behaviour and persistence length all commensurate with highly flexible coil behaviour and unlike the previously characterised tetanus toxoid protein (slightly extended and hydrodynamically compact structure with an aspect ratio of ~3). This non-protein like behaviour clearly indicates that it is the carbohydrate component which mainly influences the physical behaviour of the glycoconjugate in solution.

Advances on Bioactive Polysaccharides from Medicinal Plants.

In recent decades, the polysaccharides from the medicinal plants have attracted a lot of attention due to their significant bioactivities, such as anti-tumor activity, antioxidant activity, anticoagulant activity, antidiabetic activity, radioprotection effect, anti-viral activity, hypolipidemic and immunomodulatory activities, which make them suitable for medicinal applications. Previous studies have also shown that medicinal plant polysaccharides are non-toxic and show no side effects. Based on these encouraging observations, most researches have been focusing on the isolation and identification of polysaccharides, as well as their bioactivities. A large number of bioactive polysaccharides with different structural features and biological effects from medicinal plants have been purified and characterized. This review provides a comprehensive summary of the most recent developments in physiochemical, structural features and biological activities of bioactive polysaccharides from a number of important medicinal plants, such as polysaccharides from Astragalus membranaceus, Dendrobium plants, Bupleurum, Cactus fruits, Acanthopanax senticosus, Angelica sinensis (Oliv.) Diels, Aloe barbadensis Miller, and Dimocarpus longan Lour. Moreover, the paper has also been focused on the applications of bioactive polysaccharides for medicinal applications. Recent studies have provided evidence that polysaccharides from medicinal plants can play a vital role in bioactivities. The contents and data will serve as a useful reference material for further investigation, production, and application of these polysaccharides in functional foods and therapeutic agents.

Characterization of Capsular Polysaccharides and Their Glycoconjugates by Hydrodynamic Methods.

Hydrodynamic methods are relevant for the characterization of carbohydrates such as capsular bacterial polysaccharides or glycoconjugates in solution. This chapter focuses on the following hydrodynamic methods: sedimentation velocity analytical ultracentrifugation (SV AUC), dynamic light scattering (DLS), sedimentation equilibrium analytical ultracentrifugation (SE AUC), size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), and capillary viscometry-intrinsic viscosity measurement. The chapter highlights the general principle of these five methods, describes experimental details, and specifies advances in the last years.

An experimental design approach to the chemical characterisation of pectin polysaccharides extracted from Cucumis melo Inodorus.

Extracted pectins have been utilised in a number of applications in both the food and pharmaceutical industries where they are generally used as gelling agents, thickeners and stabilisers, although a number of pectins have been shown to be bioactive. These functional properties will depend upon extraction conditions. A statistical experimental design approach was used to study the effects of extraction conditions pH, time and temperature on pectins extracted from Cucumis melo Inodorus. The results show that the chemical composition is very sensitive to these conditions and that this has a great influence on for example the degree of branching. Higher temperatures, lower pHs and longer extraction times lead to a loss of the more acid labile arabinofuranose residues present on the pectin side chain. The fitting of regression equations relating yield and composition to extraction conditions can therefore lead to tailor-made pectins for specific properties and/or applications.

A novel method to estimate the stiffness of carbohydrate polyelectrolyte polymers based on the ionic strength dependence of zeta potential.

Polysaccharides have received a great deal of attention from, for example, the food, cosmetic and pharmaceutical industries. Their conformations (flexibility/stiffness) span a wide range of conformational flexibilities with large hydrated volumes, these properties are important in relation to polysaccharide structure-function relationships. Perhaps the simplest parameter available to estimate the dilute solution conformation of polysaccharides is the Smidsrød-Haug stiffness parameter (B) where the stiffness of polyelectrolytes can be estimated by measuring the intrinsic viscosity at a number of different ionic strengths. In this paper we propose an alternative method for estimating the Smidsrød-Haug stiffness parameter (B) using the ionic strength dependency of zeta potential. For this purpose we have studied a number of different polysaccharides.

Impact of health claims in prebiotic-enriched breads on purchase intent, emotional response and product liking.

The impact of health claims on purchase intent, emotional response and liking has never been previously reported. In this study, prebiotic-enriched bread was used as a model functional food. Purchase intent, emotional response and liking were investigated in three phases: (1) focus groups were used to gauge consumer perception of health claims and functional foods, (2) the impact of health claims on purchase intent and emotional responses were measured using an online survey (n = 122) and (3) hedonic ratings on bread rolls presented with or without any associated claims were obtained (n = 100). A cluster analysis of the purchase intent data identified two clusters of consumers who were either receptive or non-receptive to health claims. Receptive and non-receptive consumers significantly differed in the emotions they reported with respect to the claims. The hedonic ratings did not significantly differ between the breads tasted with or without health claims.

An asymmetric and slightly dimerized structure for the tetanus toxoid protein used in glycoconjugate vaccines.

Tetanus toxoid protein has been characterized with regard oligomeric state and hydrodynamic (low-resolution) shape, important parameters with regard its use in glycoconjugate vaccines. From sedimentation velocity and sedimentation equilibrium analysis in the analytical ultracentrifuge tetanus toxoid protein is shown to be mostly monomeric in solution (~86%) with approximately 14% dimer. The relative proportions do not appear to change significantly with concentration, suggesting the two components are not in reversible equilibrium. Hydrodynamic solution conformation studies based on high precision viscometry, combined with sedimentation data show the protein to be slightly extended conformation in solution with an aspect ratio ~3. The asymmetric structure presents a greater surface area for conjugation with polysaccharide than a more globular structure, underpinning its popular choice as a conjugation protein for glycoconjugate vaccines.