Composite nano-titanium oxide-chitosan artificial skin exhibits strong wound-healing effect-an approach with anti-inflammatory and bactericidal kinetics

A composite nano-TiO2-chitosan with collagen artificial skin (NTCAS) showed promising characteristics of moderate water absorptivity (110 wt.-%), fine thickness (0.34 mm), low density (0.33 mg x mm(-3)), moderate biodegradability (<3.0%), and favorable time-dependent biodegradability (0.0521 mg x mL(-1) x h(-1) on Day 0; 0.0006 mg x mL(-1) x h(-1)on Day 3 post covering). More importantly, it showed uniquely potent bactericidal (sterilization) property with relatively large values of pseudo first-order kinetic coefficients (0.1736-0.0360 min(-1)) for TiO2 contents ranging from 2.63 x 10(-5) to 2.11 x 10(-4) mg x cm(-2). In wounded subjects, NTCAS showed a steady level of TNF-alpha, while the IL-6 level reached a peak on Day 7, although significantly lower than in the control and Duoderm groups. In the animal model, NTCAS showed better and faster recovery than the other groups, which can be attributed to the unique bactericidal effect of nano-TiO2 and immune-enhancing effect of chitosan. NTCAS is a promising artificial skin substitute and is superior to any other market product currently in use.     

高效液相色谱法测定大鼠血浆中的原儿茶酸

建立了大鼠血浆中原儿茶酸含量测定的高效液相色谱方法。采用的色谱柱为DiamondsilTM C18 柱(150 mm×4.6 mm,5 μm);流动相为乙腈-水(体积比为9∶91,用H3PO4 调pH至2.5);流速1.2 mL/min;检测波长260 nm;内标为对羟基苯甲酸。原儿茶酸的线性范围为0.050~3.20 mg/L,线性相关系数为0.9978，最低定量限为0.050 mg/L,日内和日间测定的精密度（以相对标准偏差表示）均低于7.0%,准确度（以相对误差表示）为-1.4%～2.6%；在0.050,0.40,3.20 mg/L低、中、高3个添加浓度水平下，血浆样品的提取回收率分别为83.4%,87.3%,91.1%。该方法简便,灵敏,准确,适用于大鼠体内原儿茶酸的药物动力学研究。     

高效液相色谱法测定吲哚美辛控释胶囊的血药浓度和生物利用度

采用ＯＤＳ柱,甲醇－稀磷酸溶液（７６２４）为流动相,２６０ｎｍ为检测波长,建立了测定血浆中吲哚美辛浓度的高效液相色谱法,并测定了吲哚美辛控释胶囊炎痛康的血药浓度。结果表明,血浆中吲哚美辛浓度在０．１２５～５．０ｍｇ／Ｌ范围内线性关系良好（ｒ＝０．９９９６）,检测限６２．５μｇ／Ｌ（Ｓ／Ｎ＝３１）,平均回收率为１００．４％,日内和日间ＲＳＤ均小于５％。１１位受试者单剂量口服炎痛康后的相对生物利用度为１０２．３８％。     

Enhanced absorption and growth inhibition with amino acid monoester prodrugs of floxuridine by targeting hPEPT1 transporters

A series of amino acid monoester prodrugs of floxuridine was synthesized and evaluated for the improvement of oral bioavailability and the feasibility of target drug delivery via oligopeptide transporters. All floxuridine 5'-amino acid monoester prodrugs exhibited PEPT1 affinity, with inhibition coefficients of Gly-Sar uptake (IC50) ranging from 0.7 - 2.3 mM in Caco-2 and 2.0 - 4.8 mM in AsPC-1 cells, while that of floxuridine was 7.3 mM and 6.3 mM, respectively. Caco-2 membrane permeabilities of floxuridine prodrugs (1.01 - 5.31 x 10(-6 )cm/sec) and floxuridine (0.48 x 10(-6 )cm/sec) were much higher than that of 5-FU (0.038 x 10(-6) cm/sec). MDCK cells stably transfected with the human oligopeptide transporter PEPT1 (MDCK/hPEPT1) exhibited enhanced cell growth inhibition in the presence of the prodrugs. This prodrug strategy offers great potential, not only for increased drug absorption but also for improved tumor selectivity and drug efficacy.     

Quantitative determination of BMS-378806 in human plasma and urine by high-performance liquid chromatography/tandem mass spectrometry

BMS-378806 is a human immunodeficiency virus (HIV) entry inhibitor that is being developed for the oral treatment of HIV infection. Human plasma and urine LC/MS/ MS methods have been developed and validated for the quantitation of BMS-378806. For human plasma method, methyl t-butyl ether was used to extract BMS-378806 from plasma in a 96-well format, and the organic layers were dried down and then reconstituted for the injection, while a dilute-and-shoot approach was used for human urine method in a 96-well format. Chromatographic separation was achieved isocratically on a Phenomenex C18 (2) Luna column (2 x 50 mm2, 5 microm). The mobile phase contained 60:40 v/v of 0.1% formic acid in water and ACN. Detection was by positive ion electrospray MS/MS. The standard curves ranged from 1.25 to 1000 ng/mL for the plasma assay and from 10 to 5000 ng/mL for the urine assay. The curves were fitted to a 1/x2 weighted quadratic regression model for both methods. The validation results demonstrated that both methods had satisfactory precision and accuracy across the calibration ranges. The methods were applied to the analysis of human plasma and urine samples from a single ascending dose clinical study to assess the pharmacokinetics of the drug. The pharmacokinetic analysis results indicated the absorption and disposition of the drug was rapid. The systemic exposure of BMS-378806 was generally dose proportional among the doses from 100 to 1200 mg, but not dose proportional to 1600 mg. There were modest increases in the systemic exposure when the drug was given with food or given as a solution formulation. Renal excretion was not a substantial elimination pathway of the drug. BMS378806 was safe and well tolerated over a dose range of 100-1600 mg administered as a single oral dose.     

高效液相色谱法测定地榆中金丝桃甙的含量

采用高效液相色谱法测定了地榆中金丝桃甙的含量。色谱柱为Ｓｈｉｍ－ｐａｃｋＣＬＣ－ＯＤＳ柱,以甲醇－０．０２５ｍｏｌ／Ｌ磷酸（５０５０）为流动相,检测波长３７０ｎｍ,测定组分与其它组分的色谱峰达到基线分离。加样回收率平均值为９８．７％,ＲＳＤ为１．９％。为控制中药地榆的内在质量提供了新的测定指标和可靠的方法。     

Bioreducible unimolecular micelles based on amphiphilic multiarm hyperbranched copolymers for triggered drug release

A novel type of bioreducible amphiphilic multiarm hyperbranched copolymer (H40-star-PLA-SS-PEG) based on Boltorn® H40 core, poly(l-lactide) (PLA) inner-shell, and poly(ethylene glycol) (PEG) outer-shell with disulfide-linkages between the hydrophobic and hydrophilic moieties was developed as unimolecular micelles for controlled drug release triggered by reduction. The obtained H40-star-PLA-SS-PEG was characterized in detail by nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR), gel permeation chromatography (GPC), differential scanning calorimeter (DSC), and thermal gravimetric analysis (TGA). Transmission electron microscopy (TEM) and dynamic light scattering (DLS) analyses suggested that H40-star-PLA-SS-PEG formed stable unimolecular micelles in aqueous solution with an average diameter of 19 nm. Interestingly, these micelles aggregated into large particles rapidly in response to 10 mM dithiothreitol (DTT), most likely due to shedding of the hydrophilic PEG outer-shell through reductive cleavage of the disulfide bonds. As a hydrophobic anticancer model drug, doxorubicin (DOX) was encapsulated into these reductive unimolecular micelles. In vitro release studies revealed that under the reduction-stimulus, the detachment of PEG outer-shell in DOX-loaded micelles resulted in a rapid drug release. Flow cytometry and confocal laser scanning microscopy (CLSM) measurements indicated that these DOX-loaded micelles were easily internalized by living cells. Methyl tetrazolium (MTT) assay demonstrated a markedly enhanced drug efficacy of DOX-loaded H40-star-PLA-SS-PEG micelles as compared to free DOX. All of these results show that these bioreducible unimolecular micelles are promising carriers for the triggered intracellular delivery of hydrophobic anticancer drugs.     

Measurement of albuterol in guinea pig serum by high performance liquid chromatography with fluorescence detection

A sensitive, simple and reproducible high performance liquid chromatographic method for detecting and quantifying albuterol in guinea pig serum is described. A structurally related compound, bamethan, was used as an internal standard. The method employs ion-pair extraction with di(2-ethylhexyl)phosphate followed by chromatography on a Zorbax SB C18 reversed-phase column. Fluorescence detection was used to identify the compounds of interest. The calibration curve was linear between 1 and 50 ng/mL albuterol hemisulfate salt (0.83 and 41.50 ng/mL albuterol base), and the limit of detection for a 1 mL sample was 1 ng/mL albuterol hemisulfate salt (0.83 ng/mL albuterol base). Serum levels of albuterol were quantified from guinea pigs that had received the drug by continuous subcutaneous infusion at a dose of 0.2 mg/kg/day for 1, 5 or 10 days, or 10 days followed by a 24 h washout period.     

Fluoxetine and norfluoxetine analysis by direct injection of human plasma in a column switching liquid chromatographic system

A column switching LC method is presented for the analysis of fluoxetine (FLU) and norfluoxetine (NFLU) by direct injection of human plasma using a lab-made restricted access media (RAM) column. A RAM-BSA-octadecyl silica (C-18) column (40 mm x 4.6 mm, 10 microm) is evaluated in both backflush and foreflush elution modes and coupled with a C-18 lab-made (50 mm x 4.6 mm, 3 microm) analytical column in order to perform online sample preparation. Direct injection of 100 microL of plasma samples is possible with the developed approach. In addition, reduction of sample handling is obtained when compared with traditional liquid-liquid extraction (LLE) and SPE. The total analysis time is around 20 min. A LOQ of 15 ng/mL is achieved in a concentration range of 15-500 ng/mL, allowing the therapeutic drug monitoring of clinical samples. The precision values achieved are lower than 15% for all the evaluated points with adequate recovery and accuracy. Furthermore, no matrix interferences are found in the analysis and the proposed method shows to be an adequate alternative for analysis of FLU in plasma.     

Determination of ciprofloxacin with functionalized cadmium sulfide nanoparticles as a fluorescence probe

A novel assay of ciprofloxacin with a sensitivity at the microgram level is proposed based on the measurement of enhanced fluorescence intensity signals resulting from the interaction of functionalized nano-CdS with ciprofloxacin. The CdS nanoparticles was synthesized by thioacetamide (TAA) and cadmium nitrate (Cd(NO(3))(2)) in the alkaline solution. At pH 7.4, the fluorescence signals of functionalized nano-CdS were greatly enhanced by ciprofloxacin with the increase concentration of ciprofloxacin. Linear relationship can be established between the enhanced fluorescence intensity and ciprofloxacin concentration in the range of (1.25-8.75)x10(-4) mg mL(-1) ((3.77-26.4) x 10(-4)mmol L(-1)) or (8.75-1200) x 10(-4)mg mL(-1) ((26.4-3625) x 10(-4) mmol L(-1)). The limit of detection is 7.64 x 10(-6) mg mL(-1) (2.31 x 10(-5)mmol L(-1)). Based on this, a new direct quantitative determination method for ciprofloxacin in human serum samples without separation of foreign substances was established. The contents of ciprofloxacin in human serum samples were determined with recovery of 95-105% and relative standard deviation (R.S.D.) of 1.5-2.5%. This method was proved to be very sensitive, rapid, simple and tolerance of most interfering substances.     

Swarnalin and cis-swarnalin, two new tetrahydrofuran derivatives with free radical scavenging activity, from the aerial parts of Cuscuta reflexa

The reversed-phase HPLC analysis of a methanol extract of the aerial parts of Cuscuta reflexa afforded a non-separable mixture (55 : 45) of two novel tetrahydrofuran derivatives, named swarnalin (1) and cis-swarnalin (2), and a known coumarin, 5,6,7-trimethoxycoumarin (3). The structures of the compounds were elucidated unequivocally by UV, HRFABMS and a series of 1D and 2D NMR analyses. The mixture of 1 and 2 showed significant free radical scavenging activity in the DPPH assay and the RC50 value was found to be 3.80 x 10(-4) mg mL(-1) for the mixture, compared to 2.88 x 10(-5) mg mL(-1) for the positive control, quercetin.     

反相高效液相法测定穿心莲药材及其制剂中的穿心莲内酯和脱水穿心莲内酯

 发展了穿心莲药材及其中成药中两种主要成分穿心莲内酯和脱水穿心莲内酯的反相高效液相测定方法。采用甲醇振荡提取法进行样品前处理 ,在以乙腈 水为流动相作梯度洗脱、ODS柱、检测波长为 2 2 5nm的条件下 ,穿心莲内酯和脱水穿心莲内酯在 1 5min内可达到基线分离。两种内酯在 1 0mg/L～ 1 0 0mg/L时其浓度与峰面积成良好的线性关系 ,加标回收率为 96 %～ 1 0 4 %。     

Use of glucose oxidase in a membrane reactor for gluconic acid production

This article aims at the evaluation of the catalytic performance of glucose oxidase (GO) (EC.1.1.3.4) for the glucose/gluconic acid conversion in the ultrafiltration cell type membrane reactor (MB-CSTR). The reactor was coupled with a Millipore ultrafiltration-membrane (cutoff of 100 kDa) and operated for 24 h under agitation of 100 rpm, pH 5.5, and 30 degrees C. The experimental conditions varied were the glucose concentration (2.5, 5.0, 10.0, 20.0, and 40.0 mM), the feeding rate (0.5, 1.0, 3.0, and 6.0/h), dissolved oxygen (8.0 and 16.0 mg/L), GO concentration (2.5, 5.0, 10.0, and 20.0 U(GO)/mL), and the glucose oxidase/catalase activity ratio (U(GO)/U(CAT))(1:0, 1:10, 1:20, and 1:30). A conversion yield of 80% and specific reaction rate of 40 x 10(-4) mmol/h x U(GO) were attained when the process was carried out under the following conditions: D =3.0/h, dissolved oxygen =16.0 mg/L, [G] =40 mM, and (U(GO)/U(CAT)) =1:20. A simplified model for explaining the inhibition of GO activity by hydrogen peroxide, formed during the glucose/gluconic acid conversion, was presented.     

Novel biomimetic surfactant: synthesis and micellar characteristics

Novel biomimetic surfactants based on cholesterol as the hydrophobic segment and poly[2-(methacryloyloxy)ethyl phosphorylcholine] (pMPC) as the hydrophilic segment were synthesized in the present study by atom transfer radical polymerization (ATRP) of 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) using a cholesterol-based macroinitiator. The association behavior of cholesterol-block-poly[2-(methacryloyloxy)ethyl phosphorylcholine] (Chol-pMPCs) in aqueous solution was studied by (1)H NMR spectroscopy, fluorescence probe technique, and atomic force microscopy (AFM). The (1)H NMR spectrum of the polymer in CD(3)OD showed both the cholesterol group and the phosphorylcholine group while the cholesterol group did not appeared in the (1)H NMR spectrum of the polymer in D(2)O, which implied the formation of a micelle structure. Fluorescence excitation spectra of a pyrene probe solubilized in the aggregates of Chol-pMPCs suggested the presence of a critical micelle concentration (cmc) in water. The critical micelle concentrations of the polymers CMPC10, CMPC20 and CMPC40 were determined to be 7.27 x 10(-3), 13.47 x 10(-3), and 20.77 x 10(-3) mg . mL(-1), respectively. AFM images of the aggregates on mica suggested that the pMPC block formed the biocompatible micelle coronas and the cholesterol block formed the hydrophobic micelle cores. These new biomimetic diblock copolymers were evaluated as "stealthy" nanocapsules for the delivery of hydrophobic drugs. The anti-cancer drug adriamycin (ADR) was chosen as a hydrophobic drug to be incorporated into the inner core of the micelles and the morphology of the drug-loaded micelles were observed by AFM.     

Potential of various archae- and eubacterial strains as industrial polyhydroxyalkanoate producers from whey

Three different microbial wild-type strains are compared with respect to their potential as industrial scale polyhydroxyalkanoate (PHA) producers from the feed stock whey lactose. The halophilic archaeon Haloferax mediterranei as well as two eubacterial strains (Pseudomonas hydrogenovora and Hydrogenophaga pseudoflava) are investigated. H. mediterranei accumulated 50 wt.-% of poly-3-(hydroxybutyrate-co-8%-hydroxyvalerate) from hydrolyzed whey without addition of 3-hydroxyvalerate (3HV) precursors (specific productivity q(p): 9.1 mg x g(-1) x h(-1)). Using P. hydrogenovora, the final percentage of poly-3-hydroxybutyrate (PHB) amounted to 12 wt.-% (q(p): 2.9 mg x g(-1) x h(-1)). With H. pseudoflava, it was possible to reach 40 wt.-% P-3(HB-co-5%-HV) on non-hydrolyzed whey lactose plus addition of valeric acid as 3HV precursor (q(p): 12.5 mg x g(-1) x h(-1)). A detailed characterization of the isolated biopolyesters and an evaluation with regard to the economic feasibility completes the study.     

Protolytic reactions of 3,3'-dimethylnaphthidine and 3,3'-dimethoxybenzidine

The dissociation constants of diprotonated 3,3'-dimethylnaphthidine (DMN) and 3,3'-dimethoxybenzidine (DMB) have been determined spectrophotometrically. They are: pK(a1) = 2.62 +/- 0.03, pK(a2) = 3.33 +/- 0.09 for DMN: pK(a1) = 2.83 +/- 0.07, pK(a2) = 4.05 +/- 0.12 for DMB. The molar absorptivities (l.mole(-1).cm(-1)) of all forms of the indicators have been also determined: epsilon(B) = 1.68 x 10(4), epsilon(BH(+)) = 9.34 x 10(3), epsilon(BH(2+)(2)) = 1.80 x 10(3) at 300 nm for DMB; epsilon(B) = 7.33 x 10(3), epsilon(BH(+)) = 3.73 x 10(3), epsilon(BH(2+)(2)) = 0 at 330 nm for DMN.     

Immunosensor for the determination of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> using a tyrosinase–mercaptopropionic acid modified electrode as an amperometric transducer

An amperometric immunosensor for the quantification of Staphylococcus aureus based on the coimmobilization of rabbit immunoglobulin G (RbIgG) and tyrosinase on a mercaptopropionic acid self-assembled monolayer modified gold electrode is reported. A competitive mode in which protein-A-bearing S. aureus cells and antiRbIgG labeled with alkaline phosphatase (AP) compete for the binding sites of immobilized RbIgG was used. Monitoring of the affinity reaction was carried out by the amperometric detection at -0.15 V of phenol generated in the enzyme reaction with AP, at the tyrosinase-modified electrode through the electrochemical reduction of the o-quinone formed. Optimization of the working variables, such as the immunosensor composition and incubation times, the applied potential, the working pH and the concentration of phenyl phosphate used as the AP substrate, was carried out. Under the optimized conditions, both the repeatability of the measurements and the reproducibility of the responses obtained with different immunosensors yielded relative standard deviation values for the steady-state current lower than 10%. The immunosensor showed a dynamic range from 4.4x10(5) to 1.8x10(7) S. aureus cells mL(-1), with a detection limit of 1.7x10(5) cells mL(-1). The limit of detection was remarkably improved by subjecting S. aureus cells to wall lysis by heat treatment. The value obtained was 2.3x10(3) cells mL(-1), which is adequate for the monitoring of S. aureus contamination levels in some foodstuffs. As an application, milk samples spiked with bacteria at the 4.8x10(3) cells mL(-1) level were analyzed.     

Application of derivative spectrophotometry to simultaneous determination of indomethacin and 5-methoxy-2-methyl-3-indoleacetic acid in metindol injections

Derivative spectrophotometry was employed to develop a rapid and accurate method for simultaneous determination of indomethacin and 5-methoxy-2-methyl-3-indoleacetic acid as its possible impurity in Metindol injections. At the selected wavelengths, 233.04 and 284.65 nm, no interference between the components determined was observed. Under the established experimental conditions, recoveries of the particular components were from 96.14 to 98.17%. Linearity was maintained over a broad range of concentrations, from 11.88 x 10(-3) to 35.64 x 10(-3) mg/mL for indomethacin and 0.4 to 1.2 mg/mL for 5-methoxy-2-methyl-3-indoleacetic acid. The limit of detection was found to be 6.0 x 10(-3) mg/mL for indomethacin and 0.04 x 10(-3) mg/mL for 5-methoxy-2-methyl-3-indoleacetic acid. The limits of quantitation were found to be 10.0 x 10(-3) mg/mL and 0.20 x 10(-3) mg/mL, respectively.     

Encapsulation and controlled release of a hydrophobic drug using a novel nanoparticle-forming hyperbranched polyester

An amphiphilic, hyperbranched polymer suitable for use in controlled drug delivery is reported. This polymer was obtained by modification of the hyperbranched aliphatic polyester Boltorn H20 (H20) with succinic anhydride and then glycidyl methacrylate, and formed nanoparticles in aqueous solution. The critical association concentration was 7.4 x 10(-3) g . L(-1), as determined by fluorescence spectroscopy using pyrene as a molecular probe. A static/dynamic laser light scattering (LLS) study revealed that the average particle size was 39.4 nm with a low particle size distribution (PDI=0.04), and that each particle was composed of about 350 amphiphilic molecules. Daidzein, a hydrophobic traditional Chinese medicine, was encapsulated during particle formation and the release properties were determined. The optimal feeding concentration of daidzein to hyperbranched polyester was 4.9 x 10(-5) g . mL(-1) to 5.0 x 10(-3) g . mL(-1) with a loading efficiency of 76.1%. In the presence of the enzyme Lipase PS, the drug loaded nanoparticles degraded in a random one-by-one manner and released the drug over a few days. This system is therefore a novel controlled drug release system based on nanoparticles formed of hyperbranched polyester. Encapsulation of daidzein by hyperbranched polyester particles.     

Assay of naproxen by high-performance liquid chromatography and identification of its photoproducts by LC-ESI MS

A rapid, accurate and reliable reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of naproxen and its photodegradation products in methanol was developed and validated. An Inertsil 5-ODS-3V column (5 microm, C18, 250 x 4.6 mm i.d.) was used with a mobile phase of acetonitrile-methanol-1% HOAc in H2O (40:20:40, v/v/v). UV detection was set at 230 nm. The developed method satisfies system suitability criteria, peak integrity and resolution for the parent drug and its photoproducts. The intraday and interday standard deviations of five replicate determinations for five consecutive days at the working concentrations of 5.0, 10, 25, 50, and 100 microm were 0.23-0.98 with coefficients of variance (CVs) of between 0.96 and 4.56% for the former, and 0.14-1.15 with CVs of between 1.13 and 3.82% for the latter. The percentage recoveries were determined to be 98.34, 99.19, 100.18, 102.97 and 99.81%, respectively, at the five concentrations between 5.0 and 100 microm. The limit of quantitation of naproxen was determined to be 0.29 microg/mL, while the detection limit was 64 ng/mL. Four major photoproducts were observed from the HPLC chromatogram using a Panchum PR-2000 reactor which equipped with 8 W x 16 low-pressure quartz mercury lamps as the light source for irradiation of a naproxen sample in methanol. The structures of the photoproducts were confirmed by LC-ESI MS.