Octenyl-succinylated inulins for the delivery of hydrophobic drug.

The efficacy of hydrophobic anticancer drugs is limited by their poor solubility in water, inefficient target delivery, and toxic side effects. In this work, doxorubicin (DOX) was solubilized using OSA-inulins which created micellar aggregates in aqueous solution above a critical concentration. In vitro delivery of OSA-inulin-DOX micelles resulted in strong inhibition of the growth of MCF-7 breast cancer cells as compared to free DOX. They also displayed a faster cellular uptake rate, indicating that the micelles were promptly internalized into the cells through CD44 receptor-mediated endocytosis. During in vivo tumor suppression experiments in tumor-bearing mice, the OSA-inulin-DOX micelles strongly hindered tumor growth and showed substantially lower systemic toxicity compared with free DOX. Our achievements demonstrate that OSA-inulin has great potential for the encapsulating, dissolving, and targeted delivery of hydrophobic drugs, especially antitumor drugs, for nutraceutical, medical, and pharmaceutical applications.

An experimental and theoretical charge density study of theophylline and malonic acid cocrystallization.

The pharmaceutical agent theophylline (THEO) is primarily used as a bronchodilator and is commercially available in both tablet and liquid dosage forms. THEO is highly hygroscopic, reducing its stability, overall shelf-life, and therefore usage as a drug. THEO and dicarboxylic acid cocrystals were designed by Trask et al. in an attempt to decrease the hygroscopic behaviour of THEO; cocrystallisation of THEO with malonic acid (MA) did not improve the hygroscopic stability of THEO in simulated atmospheric humidity testing. The current study employed high-resolution X-ray crystallography, and Density Functional Theory (DFT) calculations to examine the electron density distribution (EDD) changes between the cocrystal and its individual components. The EED changes identified the reasons why the THEO:MA cocrystal did not alter the hygroscopic profile of THEO. The cocrystal was equally porous, with atomic packing factors (APF) similar to those of THEO 0.73 vs. 0.71, respectively. The THEO:MA (1) cocrystal structure is held together by an array of interactions; a heterogeneous synthon between the imidazole and a carboxylic fragment stabilising the asymmetric unit, a pyrimidine-imidazole homosynthon, and an aromatic cycle stack between two THEO moieties have been identified, providing 9.7-12.9 kJ mol-1 of stability. These factors did not change the overall relative stability of the cocrystal relative to its individual THEO and MA components, as shown by cocrystal (1) and THEO being equally stable, with calculated lattice energies within 2.5 kJ mol-1 of one other. The hydrogen bond analysis and fragmented atomic charge analysis highlighted that the formation of (1) combined both the EDD of THEO and MA with no net chemical change, suggesting that the reverse reaction - (1) back to THEO and MA - is of equal potential, ultimately producing THEO hydrate formation, in agreement with the work of Trask et al. These results highlight that a review of the EDD change associated with a chemical reaction can aid in understanding cocrystal design. In addition, they indicate that cocrystal design requires further investigation before becoming a reliable process, with particular emphasis on identifying the appropriate balance of synthon engineering, weak interactions, and packing dynamics.

Characterization of the polysaccharide from Cola millenii seeds.

We have isolated and characterized a water soluble polysaccharide from Cola millenii seeds. It was found to be composed of a total of 59% neutral sugars (mainly rhamnose, galactose and arabinose ~24, 13 and 8% respectively) and 41% uronic acids [mainly galacturonic acid]. The weight and number average molar mass values were found to be 4.7 × 106 g/mol and 3.5 × 106 g/mol, respectively. The polysaccharide exhibited polyelectrolyte properties with the intrinsic viscosity varying with salt concentration. The polysaccharide formed a highly viscous solution in water with apparent zero shear viscosities of 0.59-772 Pa·s at concentrations 0.3-2.5 wt%. The solutions were shear thinning even at very low concentrations. The mechanical spectra showed gel-like characteristics at concentrations >2 wt%. The rheological behavior indicates the polysaccharide has potential for application as a thickener and suspending agent in food, pharmaceutical and cosmetic formulations.

Octenyl-succinylated inulin for the encapsulation and release of hydrophobic compounds.

Octenyl-succinylated inulins (OSA-inulin) were synthesized in aqueous solutions using inulin with varying degrees of polymerization (DP). They were characterized using 1H NMR and FTIR and their degrees of substitution were determined. All the samples formed micellar aggregates in aqueous solution above a critical aggregation concentration (CAC) and solubilized beta-carotene. The amount of beta carotene solubilized within the micelles ranged from 12 to 25 mg/g of OSA-inulin and depended on the inulin molar mass. Dynamic light scattering showed that the aggregates, with and without dissolved beta-carotene, were ∼10-15 nm in size and this was confirmed by Transmission Electron Microscopy which also indicated that the micelles had a globular shape. OSA-inulin particles containing encapsulated beta-carotene were produced by freeze-drying. The encapsulated beta-carotene was not released from the freeze-dried particles when introduced into simulated gastric fluid at pH 2.5 but was readily released in simulated small intestinal fluid at pH 7. The results demonstrate the potential application of OSA-inulin in the encapsulation, dissolution and targeted delivery of hydrophobic drug molecules for nutraceutical, pharmaceutical and medical applications.

A vector holographic optical trap.

The invention of optical tweezers almost forty years ago has triggered applications spanning multiple disciplines and has also found its way into commercial products. A major breakthrough came with the invention of holographic optical tweezers (HOTs), allowing simultaneous manipulation of many particles, traditionally done with arrays of scalar beams. Here we demonstrate a vector HOT with arrays of digitally controlled Higher-Order Poincaré Sphere (HOPS) beams. We employ a simple set-up using a spatial light modulator and show that each beam in the array can be manipulated independently and set to an arbitrary HOPS state, including replicating traditional scalar beam HOTs. We demonstrate trapping and tweezing with customized arrays of HOPS beams comprising scalar orbital angular momentum and cylindrical vector beams, including radially and azimuthally polarized beams simultaneously in the same trap. Our approach is general enough to be easily extended to arbitrary vector beams, could be implemented with fast refresh rates and will be of interest to the structured light and optical manipulation communities alike.

Exploring the Binding of Barbital to a Synthetic Macrocyclic Receptor. A Charge Density Study.

Experimental charge density distribution studies, complemented by quantum mechanical theoretical calculations, of a host-guest system composed of a macrocycle (1) and barbital (2) in a 1:1 ratio (3) have been carried out via high-resolution single-crystal X-ray diffraction. The data were modeled using the conventional multipole model of electron density according to the Hansen-Coppens formalism. The asymmetric unit of macrocycle 1 contained an intraannular ethanol molecule and an extraannular acetonitrile molecule, and the asymmetric unit of 3 also contained an intraannular ethanol molecule. Visual comparison of the conformations of the macrocyclic ring shows the rotation by 180° of an amide bond attributed to competitive hydrogen bonding. It was found that the intraannular and extraannular molecules inside were orientated to maximize the number of hydrogen bonds present, with the presence of barbital in 3 resulting in the greatest stabilization. Hydrogen bonds ranging in strength from 4 to 70 kJ mol-1 were the main stabilizing force. Further analysis of the electrostatic potential among 1, 2, and 3 showed significant charge redistribution when cocrystallization occurred, which was further confirmed by a comparison of atomic charges. The findings presented herein introduce the possibility of high-resolution X-ray crystallography playing a more prominent role in the drug design process.

Characterisation and solution properties of a galactomannan from Bauhinia monandra seeds.

This study reports on the chemical and physicochemical properties of the polysaccharide isolated from Bauhinia monandra seeds. The seeds were found to contain 17.8% polysaccharide which consisted predominantly of galactose and mannose. The Man/Gal ratio was found to be approximately 4:1and the average molar mass was 2.54×105g/mol. The extracted material was also found to contain a small amount of protein (5.35%). The galactomannan produced highly viscous solution; the viscosity-shear rate profile was best described by the Williamson model. The mechanical spectrum of a 0.5wt% solution showed that G″ was greater than G' over the frequency range employed while at higher concentrations G' became greater than G″ above a critical frequency. The solutions obeyed the Cox-Merz rule at low concentrations, but there was some deviation at higher concentrations. Viscosity measurements were undertaken over a range of temperatures and the activation energy of viscous flow was found to be 20.75kJ/mol. The rheological properties of solutions of B. monandra galactomannan indicate that it has comparable characteristics to other commercially important galactomannans such as guar gum and locust bean gum and hence has potential as a thickener in the formulation of food and other related products.

Niosomes Consisting of Tween-60 and Cholesterol Improve the Chemical Stability and Antioxidant Activity of (-)-Epigallocatechin Gallate under Intestinal Tract Conditions.

In order to improve the chemical stability and antioxidant activity of (-)-epigallocatechin gallate (EGCG) in the gastrointestinal tract, niosomes composed of Tween-60 and cholesterol were developed to encapsulate EGCG in this investigation. EGCG loaded niosomes with encapsulation efficiency around 76% exhibited a small Z-average diameter about 60 nm. Compared to free EGCG, the EGCG remaining in dialysis tubes was significantly improved for niosomes at pH 2 and 7.4. Meanwhile, the residual EGCG for niosomes increased from 3% to 49% after 2 h incubation in simulated intestinal fluid (SIF). Pancreatin was found to impact the stability of niosomes in SIF mainly. Furthermore, the results from ferric reducing antioxidant power and cellular antioxidant activity tests indicated that EGCG loaded niosomes exhibited stronger antioxidant ability than free EGCG during intestinal digestion. Thus, we can infer that niosomal encapsulation might be a promising approach to improve the oral bioavailability of EGCG in the body.

A comparison of the experimental and theoretical charge density distributions in two polymorphic modifications of piroxicam.

Experimental charge density distribution studies of two polymorphic forms of piroxicam, ß-piroxicam (1) and piroxicam monohydrate (2), were carried out via high-resolution single crystal X-ray diffraction experiments and multipole refinement. The asymmetric unit of (2) consists of two discrete piroxicam molecules, (2a) and (2b), and two water molecules. Geometry differs between (1) and (2) due to the zwitterionic nature of (2) which results in the rotation of the pyridine ring around the C(10)-N(2) bond by approximately 180°. Consequently, the pyridine and amide are no longer co-planar and (2) forms two exclusive, strong hydrogen bonds, H(3)O(4) and H(2)O(3), with bond energies of 66.14 kJ mol-1 and 112.82 kJ mol-1 for (2a), and 58.35 kJ mol-1 and 159.51 kJ mol-1 for (2b), respectively. Proton transfer between O(3) and N(3) in (2) results in significant differences in surface electrostatic potentials. This is clarified by the calculation of atomic charges in the zwitterion that shows the formally positive charge of the pyridyl nitrogen which is redistributed over the whole of the pyridine ring instead of concentrating at N-H. Similarly, the negative charge of the oxygen is distributed across the benzothiazine carboxamide moiety. The multipole derived lattice energy for (1) is -304 kJ mol-1 and that for (2) is -571 kJ mol-1, which is in agreement with the experimentally determined observations of higher solubility and dissolution rates of (1) compared to (2).

Self-assembly and emulsification properties of hydrophobically modified inulin.

A series of alkenylated inulin samples were synthesized in aqueous solution using alkenyl succinic anhydrides with varying alkenyl chain lengths (C8-C18). The inulin derivatives (ASA-inulins) were characterized using NMR and FTIR and their degree of substitution determined. The solution properties of ASA-inulins were investigated using dye solubilization, surface tension, and dynamic light scattering, and all three techniques confirmed that the molecules aggregated in solution above a critical concentration (critical aggregation concentration, CAC). The value of the CAC was found to be reasonably consistent between the different techniques and was shown to decrease with increasing alkenyl chain length, from 0.08% for the octenyl succinylated sample to 0.005% for the octadecenyl succinylated sample. The hydrodynamic diameter of ASA-inulins above the CAC was determined from dynamic light scattering studies and was shown to increase with alkenyl chain length, from 4 nm for the octenyl derivative to 55 nm for the hexadecenyl derivative. All ASA-inulins were shown to be able to produce oil-in-water emulsions with a droplet size similar to that of emulsions prepared using Tween 20 on storing for 21 days. The fact that the derivatives are able to form micellarlike aggregates and stabilize emulsions makes them suitable candidates for the encapsulation and delivery of water-insoluble active compounds, with potential application in food, cosmetic, personal care, and pharmaceutical formulations.

Hydrodynamic and rheological properties of Irvingia gabonensis gum.

The polysaccharide component of Irvingia gabonensis endosperm was isolated and its hydrodynamic and rheological properties investigated. The polysaccharide is an arabinogalactan and contains small amounts of rhamnose, galacturonic acid, glucose and glucuronic acid. The polysaccharide displayed typical polyelectrolyte behaviour in solution. The intrinsic viscosity at infinite ionic strength, a measure of the hydrodynamic volume of the uncharged polysaccharide molecule, was obtained as 4.9 dl/g. The macromolecules have a semi flexible backbone with a Smidsrod stiffness parameter of 0.085. The polysaccharide exhibited non-Newtonian behaviour at all the concentrations (0.2% to 3.0% (w/v)) investigated. Cox-Merz plots showed that η(γ˙) and η(ω) were closely superimposable except at low shear rates and higher concentrations, where η>η. The high Mw (1.56 × 10(6)g/mol) and its random coil conformation show I. gabonensis polysaccharide has potential for application as a thickener.

The tachykinin peptide neurokinin B binds copper(I) and silver(I) and undergoes quasi-reversible electrochemistry: towards a new function for the peptide in the brain.

The tachykinin neuropeptide family, which includes substance P and neurokinin B, is involved in a wide array of biological functions. Among these is the ability to protect against the neurotoxic processes in Alzheimer's Disease, but the mechanisms driving neuroprotection remain unclear. Dysregulation of metal ions, particularly copper, iron and zinc is a common feature of Alzheimer's Disease, and other amyloidogenic disorders. Copper is known to be released from neurons and recent work has shown that some tachykinins can bind Cu(II) ions, and that neurokinin B can inhibit copper uptake into astrocytes. We have now examined whether neurokinin B is capable of binding Cu(I), which is predicted to be available in the synapse. Using a combination of spectroscopic techniques including cyclic voltammetry and magnetic resonance we show that neurokinin B can bind Cu(I) either directly from added CuCl or by reduction of Cu(II)-bound neurokinin B. The results showed that the Cu(I) binding site differs greatly to that of Cu(II) and involves thioether coordination via Met2 and Met10 and an imidazole nitrogen ligand from His3. The Cu(I) coordination is also different to the site adopted by Ag(I). During changes in oxidation state, copper remains bound to neurokinin B despite large changes to the inner coordination sphere. We predict that neurokinin B may be involved in synaptic copper homeostasis.

Small strain deformation measurements of konjac glucomannan solutions and the influence of borate cross-linking.

The dynamic rheology of aqueous solutions of konjac glucomannan has been evaluated over a range of concentrations up to 2.35%, and the effect of borate cross-linking of such solutions evaluated in the range 0.02-40 mM borate. In preliminary work, conventional parallel plate geometries were employed and in situ cross-linking was investigated. For borate cross-linked samples a superior method, however, was found to be measurement of pre-formed cores of cross-linked polymer into which a four-bladed vane geometry was introduced. In order to compare with other associating polymer systems, rheological data were analysed by defining plateau moduli, corresponding relaxation times and zero shear viscosities and the scaling behaviour of these parameters with polymer and cross-linker concentrations was established. Maxwell fits and time-concentration superposition procedures were investigated. The rheological properties of the cross-linked polymer were shown to be the result of both increased network connectivity and retarded network dynamics.

Control of the properties of xanthan/glucomannan mixed gels by varying xanthan fine structure.

The interaction of native xanthan gum, deacetylated xanthan gum and depyruvated xanthan gum with konjac glucomannan has been studied using DSC and controlled stress rheometry. In the absence of electrolyte the DSC cooling curves for native xanthan and deacetylated xanthan showed a single peak and there was a corresponding sharp increase in the storage modulus indicating gel formation. It is apparent that on cooling, association of the konjac glucomannan with the native xanthan molecules is triggered by the xanthan coil-helix transition. In the presence of electrolyte, there were two DSC peaks observed. The higher temperature DSC peak was attributed to the xanthan coil-helix transition while the lower temperature DSC peak was attributed to konjac glucomannan-xanthan association as noted by an increase in the storage modulus. The gels formed were much weaker than those in the absence of electrolyte. The DSC cooling curves for depyruvated xanthan in the absence of electrolyte showed two peaks. The higher temperature peak was attributed to the coil-helix transition while the lower temperature peak corresponded to gelation as noted by an increase in the storage modulus. The gels were very much weaker than for native xanthan gum and deacetylated xanthan gum.

Rheological characterization of the galactomannan from Leucaena leucocephala seed.

A water soluble galactomannan isolated from Leucaena leucocephala seeds gave an intrinsic viscosity of 3.5 dl/g and viscosity average molecular mass, M(v), of 6.98 × 10(5) g/mol. This was in reasonably good agreement with the value of the weight average molecular mass, M(w), of 5.44 ± 0.20 × 10(5) g/mol determined by GPC-MALLS coupled to RI. The onset of polymer coil overlap occurred at c*[η] of 2.1, with slope of 3.0 above and 1.3 below the point of polymer coil overlap. The shear viscosity of the polysaccharide was temperature dependent and decreased with increasing temperature. The activation energy for viscous flow of 3.0% polysaccharide concentration obtained by Arrhenius plot of zero shear viscosity as a function of temperature was 26.4 kJ/mol. Both the storage modulus (G') and loss modulus (G″) showed strong dependence on frequency indicating the presence of entangled coils. The Cox-Merz plot gave close superimposition of the complex and shear viscosities.

MhbT is a specific transporter for 3-hydroxybenzoate uptake by Gram-negative bacteria.

Klebsiella pneumoniae M5a1 is capable of utilizing 3-hydroxybenzoate via gentisate, and the 6.3-kb gene cluster mhbRTDHIM conferred the ability to grow on 3-hydroxybenzoate to Escherichia coli and Pseudomonas putida PaW340. Four of the six genes (mhbDHIM) encode enzymes converting 3-hydroxybenzoate to pyruvate and fumarate via gentisate. MhbR is a gene activator, and MhbT is a hypothetical protein belonging to the transporter of the aromatic acid/H(+) symporter family. Since a transporter for 3-hydrxybenzoate uptake has not been characterized to date, we investigated whether MhbT is responsible for the uptake of 3-hydroxybenzoate, its metabolic intermediate gentisate, or both. The MhbT-green fluorescent protein (GFP) fusion protein was located on the cytoplasmic membrane. P. putida PaW340 containing mhbRΔTDHIM could not grow on 3-hydroxybenzoate; however, supplying mhbT in trans allowed the bacterium to grow on the substrate. K. pneumoniae M5a1 and P. putida PaW340 containing recombinant MhbT transported (14)C-labeled 3-hydroxybenzoate but not (14)C-labeled gentisate and benzoate into the cells. Site-directed mutagenesis of two conserved amino acid residues (Asp-82 and Asp-314) and a less-conserved residue (Val-311) among the members of the symporter family in the hydrophilic cytoplasmic loops resulted in the loss of 3-hydroxybenzoate uptake by P. putida PaW340 carrying the mutant proteins. Hence, we demonstrated that MhbT is a specific 3-hydroxybenzoate transporter.

Status assessment of New Zealand's naturally uncommon ecosystems.

Globally, ecosystems are under increasing anthropogenic pressure; thus, many are at risk of elimination. This situation has led the International Union for Conservation of Nature (IUCN) to propose a quantitative approach to ecosystem-risk assessment. However, there is a need for their proposed criteria to be evaluated through practical examples spanning a diverse range of ecosystems and scales. We applied the IUCN's ecosystem red-list criteria, which are based on changes in extent of ecosystems and reductions in ecosystem processes, to New Zealand's 72 naturally uncommon ecosystems. We aimed to test the applicability of the proposed criteria to ecosystems that are naturally uncommon (i.e., those that would naturally occur over a small area in the absence of human activity) and to provide information on the probability of ecosystem elimination so that conservation priorities might be set. We also tested the hypothesis that naturally uncommon ecosystems classified as threatened on the basis of IUCN Red List criteria contain more threatened plant species than those classified as nonthreatened. We identified 18 critically endangered, 17 endangered, and 10 vulnerable ecosystems. We estimated that naturally uncommon ecosystems contained 145 (85%) of mainland New Zealand's taxonomically distinct nationally critical, nationally endangered, and nationally vulnerable plant species, 66 (46%) of which were endemic to naturally uncommon ecosystems. We estimated there was a greater number of threatened plant species (per unit area) in critically endangered ecosystems than in ecosystems classified as nonthreatened. With their high levels of endemism and rapid and relatively well-documented history of anthropogenic change, New Zealand's naturally uncommon ecosystems provide an excellent case-study for the ongoing development of international criteria for threatened ecosystems. We suggest that interactions and synergies among decline in area, decline in function, and the scale of application of the criteria be used to improve the IUCN criteria for threatened ecosystems.

A kinetic analysis of three modified novel nitroreductases.

A kinetic comparison between three nitroreductase enzymes isolated from the genome of Bacillus licheniformis ATCC 14580 for prospective use as immobilised enzymes for explosives detection has been conducted. The genes encoding the three enzymes (yfkO [BLNfnB] encoding an NfsB-like enzyme; nfrA [BLNfrA1] and ycnD [BLNfrA2] encoding PnrA-like enzymes) have been PCR amplified from the native genome and cloned into pET-28a(+) and a modified cysteine((6))-tagged pET-28a(+) and subsequently over-expressed, purified, and biochemically characterised. The previously uncharacterised nitroreductases exhibited activity against a wide range of explosives, including cyclic nitramines. Amino acid alignments and overall structural comparisons with other nitroreductase family members suggest that the B. licheniformis enzymes are members of the NfsA-Frp/NfsB-FRase I family group. Despite the overall low amino acid identity, regions for flavin mononucleotide binding and active site residues were highly conserved.

Antimony biomethylation in culture media revisited in the light of solubility and chemical speciation considerations.

Laboratory culture experiments have shown that antimony biomethylation can result from bacterial and fungal activity under both aerobic and anaerobic conditions. However, in the light of our current knowledge of antimony solubility and equilibria, critical analysis of the conditions used in published laboratory studies reveals that solution chemistry was generally overlooked and oversaturated solutions were used. As a result, it is very difficult, if not impossible, to establish reliable observed effect-concentration relationships in the experiments published.

Interaction of gum arabic with fatty acid studied using electron paramagnetic resonance.

Electron paramagnetic resonance (EPR) is here used to study the interaction between gum arabic and a fatty acid. The EPR spectra of 5-doxyl stearic acid (5-DSA), a spin-labeled fatty acid analog, displayed increasingly anisotropic line features upon addition of gum arabic, indicating a strong immobilization of the nitroxyl moiety when the fatty acid is bound to gum arabic. To understand the nature of the interaction, EPR measurements were carried out at different pHs and using two fractions of gum arabic separated by hydrophobic interaction chromatography (HIC). 5-DSA bound favorably to the hydrophobic fraction, which contains mainly glycoprotein, and a small amount of high molecular weight arabinogalactan protein (AGP). Binding occurred to a less extent to the hydrophilic fraction, which contains essentially arabinogalactan (AG). Such a hydrophobic binding mechanism is further supported by a sharp drop in the binding when pH is raised above the pK(a) value of 5-DSA (approximately pH 5). This is because the ionization of carboxylic groups would lead to increased polarity and hydrophilicity of the fatty acid. A secondary effect involving the formation of ionic hydrogen bonds between carboxylic groups in fatty acid and lysine residues in gum arabic might also contribute. This is consistent with the reduction in binding ability when the pH was elevated above the pK(a) value of lysine residue (approximately pH 10). The biological significance of these findings is considered.