Properties of calcium-induced gel beads prepared with alginate and hydrolysates

Calcium-induced alginate gel beads (Alg-Ca) containing alginate hydrolysate, such as the guluronic acid block (GB), was prepared and the drug release profiles were investigated under simulated gastrointestinal conditions. The addition of GB to Alg-Ca altered its rheological properties. A model drug (hydrocortisone) was incorporated at 78% of its theoretical yield within the dried Alg-Ca containing 5% GB and it was gradually released from the beads in JP XIV 1st medium for disintegration test (pH 1.2), while it was rapidly released with disintegration of the gel matrix in JP XIV 2nd medium (pH 6.8). In contrast, for Alg-Ca containing GB and chitosan, disintegration was not observed in these media and the drug release rate was markedly different. These results demonstrate that the release profiles of drugs incorporated into Alg-Ca can be controlled by adding these polysaccharides.     

Effects of natural polysaccharide addition on drug release from calcium-induced alginate gel beads

Calcium-induced alginate gel beads (Alg-Ca) containing various polysaccharides, including an alginate hydrolysate, were prepared and the drug release profiles were investigated. Hydrocortisone (HC) was gradually released from Alg-Ca into the mimic gastric fluid, while in intestinal fluid, it was quickly released with the dissolution of Alg-Ca. However, with Alg-Ca containing 5% chitin (CT), dissolution of Alg-Ca was not observed, and release of HC showed apparent zero-order kinetics. Furthermore, addition of the alginate hydrolysate altered the HC-release profile for Alg-Ca.     

The controlled release of a drug from biodegradable chitosan gel beads

Chitosan (CS) forms a gel in solutions with a pH above 12, and the gelation occurs at pH of about 9 in 10% amino acid solutions. In this paper, we investigated the enzymatic degradation and the drug release profile of this novel CS gel beads. The degradability of the CS gel beads was affected by the CS properties, e.g. the degree of deacetylation. The release of prednisolone (PS), as a model drug, from the CS gel beads was sustained significantly compared with the gel prepared with NaOH only. However, the release was not able to be sustained by the increment of NaOH concentration in the solution employed for the preparation of CS gel beads. We also investigated the control of drug release from CS gel beads by application of a complex formed between chondroitin sulfate (Cho) and CS. The release of PS from the CS gel beads treated with Cho was prolonged, and the release pattern was not affected by the treatment time. The time to 50% drug release was about 5 min with PS powder, about 200 min in CS gel beads with 10% glycine (Gly) (pH 9.0), and about 330 min in the CS gel beads with 10% Gly (pH 9.0) treated with Cho. Thus CS gel beads appear promising as a vehicle for sustained drug delivery, and the degradation of CS gel beads may be controlled by the degree of deacetylation of CS.     

The drug release profile from calcium-induced alginate gel beads coated with an alginate hydrolysate

Calcium-induced alginate gel bead (Alg-Ca) coated with an alginate hydrolysate (Alg), e.g. the guluronic acid block (GB) was prepared and the model drug, hydrocortisone release profiles were investigated under simulated gastrointestinal conditions. Their molecular weights were one sixth or one tenth that of Alg and the diffraction patterns of the hydrolysates resembled that of Alg. The drug release rate from Alg-Ca coated with GB apparently lowered than that of Alg-Ca (coating-free) in the gastric juice (pH1.2). And the coating did not resist the disintegration of Alg-Ca in the intestinal juice (pH 6.8) and the gel erosion accelerated the drug release. On the other hand, for the coated Alg-Ca containing chitosan, the drug release showed zero-order kinetics without rapid erosion of Alg-Ca. The drug release rate from Alg-Ca was able to be controlled by the coating and modifying the composition of the gel matrix.     

Preparation and drug retention of biodegradable chitosan gel beads.

Chitosan (CS) gel beads containing drug could be prepared in amino acid solutions of pH about 9, despite the requirement for a pH above 12 for gelation in water. This phenomenon was observed not only in amino acid solutions but also in solutions of compounds having amino groups. A solute concentration of more than 10% was required for preparation of gel beads at pH 9. Gelation of the CS beads required about 25 to 40 min, depending on the species of amino acid. Lidocaine hydrochloride (LC) as a model drug was retained in the beads to about 20 to 35% of the theoretical total amount, despite being a water-soluble drug. The release of LC from the CS gel beads was prolonged. The release pattern was not affected by the species of amino acid or CS, or the preparation time.     

Quantification of bile acids directly from urine by MALDI–TOF–MS

The ability to quantify mixtures of bile acids using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry directly from urine has been demonstrated. Six cholic acid derivatives were selected for analysis: taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), taurolithocholic acid (TLCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), and glycolithocholic acid (GCDCA). Urine samples were pre-concentrated and purified using solid-phase extraction (SPE) columns. The method was optimized to eliminate suppression effects, and proved to be reproducible from day to day. Calibration curves averaged from three days were obtained for the bile acids directly from urine, and then tested for their ability to accurately determine concentrations from one measurement. In summary, a simple, rapid method has been developed for the quantification of bile salts from urine with SPE clean-up by MALDI-MS.     

Novel permethylated beta-cyclodextrin derivatives appended with chromophores as efficient fluorescent sensors for the molecular recognition of bile salts

Two novel permethylated beta-cyclodextrin (PM-beta-CD) derivatives, i.e., 6I-O-(1-naphtholxy)-2I,31-di-O-methylhexakis(2II-VII,3II-VII,6II-VII-tri-O-methyl)-beta-cyclodextrin (1) and 6I-O-(8-hydroxyquinoline)-2I,31-di-O-methylhexakis(2II-VII,3II-VII,6II-VII- tri-O-methyl)-beta-cyclodextrin (2), were synthesized in satisfactory yields, and their inclusion modes, complex-induced fluorescent behaviors, binding ability, and selectivity for bile salts of biological relevance (cholic acid sodium salt, CA; deoxycholic acid sodium salt, DCA; glycochoic acid sodium salt, GCA; taurocholic acid sodium salt, TCA) were investigated by the circular dichroism, 2D NMR, steady-state, and time-resolved fluorescent spectra. The results obtained from induced circular dichroism and ROESY spectra show that the chromophore groups of 1 and 2 reside in the central cavity of PM-beta-CD, and are expelled to the region of narrow torus rim upon complexation with bile guests, which presents the binding mode of cooperative inclusion. The transfer of the chromophore groups from the central cavity to the more hydrophobic torus rim leads to the remarkable increase of fluorescent intensities and longer fluorescent lifetimes of hosts 1 and 2 upon gradual addition of bile salts, which is importantly distinct from the molecular recognition of the chromophore-modified beta-CD species with bile salts. Interestingly, hosts 1 and 2 present much stronger binding ability for bile guests than PM-beta-CD. Differing from native beta-CD, all the PM-beta-CDs are more prone to include bile salts with longer tails, such as GCA and TCA. Their corresponding binding ability and molecular selectivity are closely discussed from the viewpoints of difference of cavity size/shape between beta-CD and PM-beta-CD, effect of substituent groups, and structures of bile guests, respectively.     

A single hydroxyl group governs ligand site selectivity in human ileal bile acid binding protein

The recognition between proteins and their native ligands is fundamental to biological function. In vivo, human ileal bile acid binding protein (I-BABP) encounters a range of bile salts that vary in the number and position of steroidal hydroxyl groups and the presence and type of side-chain conjugation. Therefore, it is necessary to understand how chemical variability in the ligand affects the energetic and structural aspects of its recognition. Here we report studies of the binding site selectivity of I-BABP for glycocholic (GCA) and glycochenodeoxycholic (GCDA) acids using isotope-enriched bile salts along with two-dimensional heteronuclear NMR methods. When I-BABP is presented with either GCA or GCDA alone, the ligands bind to both sites. However, when presented with an equimolar mixture of the two bile salts, GCDA binds exclusively to site 1 and GCA to site 2. This remarkable selectivity is governed by the presence or absence of a single hydroxyl group at the C-12 position of the steroid tetracycle. The basis for this site selectivity appears to be energetic rather then steric.     

Diastereomeric molecular recognition and binding behavior of bile acids by L/D-tryptophan-modified beta-cyclodextrins

[reaction: see text] Binding behavior of L- and D-tryptophan-modified beta-cyclodextrins (L/D-Trp-beta-CD) (1 and 2) with four bile acids, i.e., cholate (CA), deoxycholate (DCA), glycocholate (GCA), and taurocholate (TCA), has been investigated by fluorescence, circular dichroism, and 2D-NMR spectroscopies and fluorescence lifetime measurement, as well as isothermal titration microcalorimetry. From the induced circular dichroism (ICD) and 2D NMR spectra, it is deduced that the D-Trp moiety of 2 attached to beta-CD is more deeply self-included in the cavity than that of the antipodal L-Trp moiety of 1, indicating appreciably enantioselective binding of the chiral sidearm by beta-CD. Interestingly, the original difference in conformation between 1 and 2 led to quite a large difference in affinity toward DCA, giving 3.3 times higher binding ability for 2 than for 1. Thermodynamically, the inclusion complexation of 1 and 2 with bile acids was entirely driven by favorable enthalpy change (DeltaH degrees) with accompanying negative entropy change (DeltaS degrees). The stronger binding of bile acids by L/D-Trp-beta-CD is attributable to higher enthalpic gains. The combined use of the calorimetric and NMR ROESY spectral examinations revealed the correlation between the thermodynamic parameters and the role of sidearm conformation in modified beta-cyclodextrins.     

A disulfide bridge allows for site-selective binding in liver bile acid binding protein thereby stabilising the orientation of key amino acid side chains

The presence of a disulfide bridge in liver bile acid binding protein (L-BABP/S-S) allows for site-selective binding of two bile acids, glycochenodeoxycholic (GCDA) and glycocholic acid (GCA), differing only in the presence of a hydroxyl group. The protein form devoid of the disulfide bridge (L-BABP) binds both bile salts without discriminating ability. We investigate the determinants of the molecular recognition process in the formation of the heterotypic L-BABP/S-S complex with GCA and GCDA located in the superficial and inner protein sites, respectively. The comparison of the NMR spectroscopy structure of heterotypic holo L-BABP/S-S, the first reported for this protein family, with that of the homotypic L-BABP complex demonstrates that the introduction of a S-S link between adjacent strands changes the conformation of three key residues, which function as hot-spot mediators of molecular discrimination. The favoured χ(1) rotameric states (t, g(+) and g(-) for E99, Q100 and E109 residues, respectively) allow the onset of an extended intramolecular hydrogen-bond network and the consequent stabilisation of the side-chain orientation of a buried histidine, which is capable of anchoring a specific ligand.     

Application of zirconium-modified silica gel as a stationary phase in the ion-exclusion chromatography of carboxylic acids I. Separation of benzenecarboxylic acids with tartaric acid as eluent and with UV-photometric detection

The application of zirconium-modified silica gels (Zr–Silicas) as stationary phases for ion-exclusion chromatography with UV-photometric detection (IEC–PD) for mono-, di-, tri- and tetrabenzenecarboxylic acids (pyromellitic, trimellitic, hemimellitic, o-phthalic, salicylic and benzoic acids) and phenol was carried out using tartaric acid as the eluent. Zr–Silicas were prepared by the reaction of the silanol group on the surface of silica gel with zirconium tetrabutoxide [Zr(OCH₂CH₂CH₂CH₃)₄] in ethanol solution. The effect of the amount of zirconium adsorbed on silica gel on chromatographic behavior of these benzenecarboxylic acids and phenol was investigated. As a result, Zr–Silica adsorbed on 20 mg zirconium g⁻¹ silica gel was the most suitable stationary phase in the IEC–PD for the simultaneous separation of these benzenecarboxylic acids and phenol. Excellent simultaneous separation and highly sensitive UV detection at 254 nm for these benzenecarboxylic acids and phenol were achieved in 20 min by the IEC–PD using the Zr–Silica column (250×4.6 mm I.D.) and a 10 mM tartaric acid at pH 2.5 as eluent.     

Application of zirconium-modified silica gel as a stationary phase in the ion-exclusion chromatography of carboxylic acids II. Separation of aliphatic carboxylic acids with pyromellitic acid as eluent and with suppressed conductimetric detection

The application of zirconium-modified silica gels (Zr-Silica) as stationary phases for ion-exclusion chromatography with conductimetric detection (IEC–CD) for C₁–C₈ aliphatic carboxylic acids (formic, acetic, propionic, butyric, valeric, caproic, heptanoic and caprylic acids) was carried out using pyromellitic acid as the eluent. Zr-Silicas were prepared by the reaction of the silanol group on the surface of silica gel with zirconium tetrabutoxide [Zr(OCH₂CH₂CH₂CH₃)₄] in ethanol solution. An ASRS-Ultra anion self-regenerating suppressor in the K⁺ form was used for the enhancement of conductimetric detector response of these aliphatic carboxylic acids. A Zr-Silica adsorbed on 10 mg zirconium g⁻¹ silica gel was the most suitable stationary phase in IEC–CD for the separation of these aliphatic carboxylic acids. Excellently simultaneous separation and highly sensitive detection for these aliphatic carboxylic acids were achieved in 25 min by IEC–CD with the Zr-Silica column (250×4.6 mm I.D.) and a 0.2 mM pyromellitic acid containing 0.15% heptanol as the eluent.     

壳聚糖-g-聚甲基丙烯酸凝胶粒的制备及其药物释放行为

以壳聚糖和甲基丙烯酸为原料,硝酸铈铵为引发剂,合成了不同接枝率的壳聚糖-g-聚甲基丙烯酸(CS-g-PMAA),用FTIR、1H NMR和元素分析表征了产物的结构,以柠檬酸三钠和戊二醛为交联剂制备了具有核壳结构的CS-g-PMAA载药体系。 用UV/Vis检测了CS-g-PMAA粒子对模型药物的释放行为。 结果表明,CS-g-PMAA接枝率为12.21%时药物释放速率最慢,其在pH=1.8介质中药物累积释放量(11 h)为44.18%,而壳聚糖粒子的累积释放量高达65.24%,即接枝改性壳聚糖粒子对药物的缓慢控制释放性能较好; CS-g-PMAA粒子的释药行为还依赖于介质的pH值和盐浓度,在低pH值和低盐浓度下,药物释放速率较快;酶环境下由于载体材料的降解使药物释放速率加快。 分析了不同条件下CS-g-PMAA载药粒子中药物的释放机理。     

Preparation of alginate coated chitosan hydrogel beads by thermosensitive internal gelation technique

Chitosan hydrogel beads were successfully prepared by the method of thermosensitive internal gelation technique. The prepared beads were spherical, smooth-surfaced and non-aggregated with a diameter of 1.7–2.1 mm. The diameters of beads can be controlled and have a correlation with the initial drop size, the concentration of CaCl₂, alginate and the time of solidification. The bead is comprised of three parts, which are chitosan/glycerophosphate (CS/GP) hydrogel core, chitosan-alginate (CS/SA) gel layer in the middle and calcium-alginate gelatin capsules in outer layer. Swelling studies indicate that the beads can be stable in simulated gastric fluid. But the beads shrink sharply when removed to simulated intestinal fluid. Drug release behavior showed that release of ornidazole in the beads is much slower than in the CS/GP hydrogel.     

Determination of critical micellar concentrations of two monoketo derivatives of cholic acid

Critical micellear concentrations (CMC) were determined for two novel promoters of membrane permeability—7-monoketocholic acid (7-MKC) and 12-monoketocholic acid (12-MKC), using two non-invasive (¹H NMR relaxation experiment and conductometry) and two invasive (spectral shift and partition coefficient of the probe molecule) methods. Studies by the former methods suggest the different aggregation abilities of the investigated bile acid derivatives. In an aqueous solution, 7-MKC has a somewhat lower CMC value (43 mM) than 12-MKC (50 mM). Further, it was found that, in addition to primary micelles, 7-MKC forms also secondary micelles. In the experiments with probe (hydrophobic) molecules, the aggregation properties of investigated bile acids did not differ in water, whereas the presence of urea altered the aggregation of 7-MKC.Based on the CMC value, 7-MKC is more hydrophobic than 12-MKC. The apparent hydrophobicity of 7-MKC is a consequence of the formation of secondary micelles, shifting the monomer equilibrium to the direction of primary micelles, which is manifested as a decrease in the CMC value.     

Swelling of poly(methacrylic acid) gels and adsorption of L-lysine and its polymer on the gels

Poly (methacrylic acid) gels (PMAA gels) of various degrees of crosslinking were prepared and the dissociation behavior of these gels was examined; the swelling behavior was investigated as a function of the solution pH values. A reentrant phenomenon of swelling was observed and interpreted based on the Flory-Huggins equation and the Donnan equilibrium formula. Moreover, adsorption of L-lysine, oligo(L-lysine)s (Lys-n,n=3, 9, and 19) and poly(L-lysine) onto PMAA gels from aqueous solutions was investigated under different conditions of pH and concentration of adsorbate. The adsorption ratio of L-lysine onto PMAA gel is dependent on both the pH of solution and the degree of crosslinking. In a pH range between 8 and 9, the protonated form of L-lysine is strongly adsorbed on the PMAA gel by electrostatic interactions. Oligomers and polymer of L-lysine are adsorbed in a somewhat different way from the monomeric L-lysine. In addition, the desorption behavior of L-lysine from PMAA gels by a change in pH was also investigated.     

Effect of organic and inorganic acids on chitosan/glycerophosphate thermosensitive hydrogel

The influence of organic and inorganic acids on chitosan/glycerophosphate (CS/GP) hydrogel has been investigated by dissolving chitosan in different acids. The results of gelation showed that all of the chitosan dissolved in monovalent acid solutions (i.e., formic, acetic, propionic, butyric, isobutyric, lactic, nitric, hydrochloric, and chloroacetic acid), when neutralized by GP solution, could transform into hydrogel after 2–5 min at 37 °C, while those dissolved in multivalent acids failed in gel formation. The inner structures of CS/GP hydrogels prepared with monovalent acids depended on the ionic strength and chain length of acids. Morphological examination by scanning electron microphotography demonstrated that large pores occurred during the gel-forming process, and the aperture size was also related to different acids. The cytotoxicity studies indicated that CS/GP systems prepared by dissolving chitosan in tested acids except chloroacetic acid were nontoxic to mouse embryonic fibroblasts and Hela cells.     

Determination of conjugated bile acids in human urine by high-performance liquid chromatography with chemiluminescence detection.

A qualitative and quantitative analysis of the conjugated 1 beta- and 6 alpha-hydroxy bile acids, including common bile acids, in human urine using high-performance liquid chromatography with chemiluminescence detection is described. After extraction of urine with C18 silica cartridges, the bile acids were separated into non-conjugated, glycine, taurine and sulphate fractions by ion-exchange chromatography on a lipophilic gel. Solvolysis of the sulphate was carried out by treatment with trifluoroethanol in acetone containing hydrochloric acid, and the liberated amino acid conjugates were fractionated again. The individual bile acids were separated on a reversed-phase C18 column (Bile Pak II), with detection by an immobilized 3 alpha-hydroxysteroid dehydrogenase enzyme reactor and chemiluminescence reaction of the generated NADH using 1-methoxy-5-methylphenazinium methylsulphate-isoluminol-microperoxidase system. The assay method showed the detection limits ranging from 8 to 250 pmol for the bile acids tested. Analysis of urine samples obtained from newborns, non-pregnant women and women in late pregnancy showed a large difference in bile acid composition and conjugation mode, suggesting that bile acid metabolism is different during fetal and neonatal periods.     

Effect of MWCNTs Doping on the Morphology,Structure and Properties of Chitosan Beads

A series of chitosan (CS)/multi-walled carbon nanotubes (MWCNTs) composite hydrogel beads with different MWCNTs contents are prepared via a solution blending method. The effects of MWCNTs on the morphology, structure and properties of chitosan beads have been investigated. Digital pictures show that the composite beads obtained are of good morphological characteristics, and the SEM micrographs indicate that the addition of MWCNTs into CS beads made the surface of the CS/MWCNTs hydrogel beads contain much larger wrinkles. Fourier transform infrared spectra (FTIR) show that the main chain of CS bead is not changed, but there are some electrostatic interactions between CS and MWCNTs, which lead to very significant changes in the crystallization behavior of CS and MWCNTs. The thermal stability of CS/MWCNTs composites at high temperatures is increased with the existence of MWCNTs, indicating a possible electrostatic interaction between MWCNTs and CS lattices to limit the motivation of CS. The adsorption capacity of CS beads doped with a lower percentage of MWCNTs (0.02 wt%) for acid fuchsin is 112.76 mg/g, higher than that of pure CS beads (35.62 mg/g).     

Development of an HPLC-fluorescence determination method for carboxylic acids related to the tricarboxylic acid cycle as a metabolome tool

We report the simultaneous determination of the carboxylic acids related to the tricarboxylic acid (TCA) cycle, which plays an important role in producing adenosine triphosphate (ATP) and generating energy in mitochondria. Seven carboxylic acids from the TCA cycle, and pyruvic acid and 2-methylsuccinic acid, as an internal standard, were derivatized with a fluorescent reagent for carboxyl groups, 4-N,N-dimethylaminosulfonyl-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ), in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 4-N,N-dimethyaminopyridine as the coupling reagents, at 60 degrees C for 120 min. Subsequently, the excess DBD-PZ was removed efficiently using a cation-exchange cartridge, SDB-RPS (Empore). These fluorescent derivatives were separated well from each other on an octadecyl silica column (TSKgel ODS-80Ts, 250 x 4.6 mm, i.d.) with an eluent of acetonitrile-water containing 1% formic acid at a flow rate of 0.8 mL/min, and were detected fluorometrically at 560 nm, with excitation at 450 nm. The validation data were satisfactory in the range of 2.5-100 microm citric acid, isocitric acid, 2-oxoglutaric acid, succinic acid and fumaric acid. The detection limit (S/N = 3) for citric acid was 2 fmol on the column. The structures of these derivatives were confirmed by high-performance liquid chromatography-mass spectrometry, which proved that their carboxylic groups were completely labeled with DBD-PZ, except for oxaloacetic acid. This HPLC method was successfully applied to the analysis of TCA cycle metabolites in rat urine. The method will also be useful for metabolome research, such as for target analyses of metabolites with carboxyl groups, not only in urine but also in cells and organs.