We developed poly lactic-co-glycolic acid (PLGA) microspheres loaded with cefquinome and tested their effectiveness in a mouse model. The microspheres were prepared by optimizing several key parameters such as PLGA molecular weight, drug/polymer ratio, internal water volume and ethyl acetate. Drug loading efficiency, stability, in vitro release and tissue distribution in mouse were evaluated. The average particle size of the microspheres was 27.84 μm. The drug loading efficiency was 64.57%. The in vitro release of cefquinome from microspheres after 4 h was about 40% compared with over 90% for the drug alone. The concentration of cefquinome in lung reached 25 μg/g 0.25 h after injection, and kept at 10 μg/g 4 h after injection. However, the concentration of cefquinome was very low in other organs even 0.25 h after injection. In conclusion, Cefquinome-loaded PLGA microspheres are compatible as an effective lung-targeting drug delivery system and have a good sustained release efficacy.

•A novel and amplified fluorescence assay for folate receptor activity was developed.•Terminal protection and exonuclease I assisted cleavage-induced DNAzyme amplification.•A detection limit of 0.2 ng/mL for folate receptor could be achieved.The folate receptor (FR) is known as an attractive tumor marker that is expressed in a variety of cancers at high frequency. Based on the specific interaction of FR toward folic acid(FA)-linked DNA, we proposed a simple, rapid and selective fluorescent method for detection of FR based on terminal protection and exonuclease I (Exo I) assisted cleavage-induced DNAzyme releasing strategy. In the present system, with the addition of FR, the digestion of Exo I cannot proceed past the FR-linked DNA, then the 8–17 DNAzyme substrate was successfully conserved and show its cyclic catalytic cleavage toward molecular beacon (MB) substrate, resulting in an evident fluorescence signal enhancement. Combining with DNAzyme as a singal amplification, this developed terminal protection and Exo I assisted cleavage platform have detection concentration of FR range from 0.2 to 40 ng/mL and achieve a detection limit of 0.2 ng/mL. Moreover, we further employed the proposed method to measure FRs in serum samples with satisfactory results. This developed terminal protection and Exo I assisted cleavage strategy combined with 8–17 DNAzyme opens a promising avenue for FR detection, and should be also conveniently used for the sensor of other proteins or nucleic acid and may have widespread applications in biological process researches, drug discovery, and clinic diagnostics.Download high-res image (87KB)Download full-size image

For the first time, the surfactant-free microemulsion reinforced hollow-fiber liquid-phase microextraction (SFME-HF-LPME) was developed for the determination of phthalic acid esters (PAEs) followed by field-enhanced sample injection with reverse migrating micelles (FESI-RMM) in capillary electrophoresis. An SFME composed of toluene, isopropanol, and water was used to reinforce HF. The prepared SFME-HF was applied to extract PAEs. After extraction, analytes were desorbed and analyzed by capillary electrophoresis. Several important parameters influencing the extraction efficiency of SFME-HF-LPME such as the type of extraction solvent, SFME component, extraction time, desorption condition, pH value, stirring rate, and salting-out effects were optimized. The proposed method provided a good linearity, low limits of detection, and good repeatability of the extractions. This method was then utilized to analyze trace PAEs in beverage and urine. The results indicated that the developed method is an excellent alternative for the PAEs routine analysis in the food and biological field.

An electrophoretically mediated microanalysis (EMMA) method for screening neuraminidase inhibitors in depolymerized glycosaminoglycan and natural extracts is described. In the present method, enzyme and substrate were individually introduced into the capillary as distinct plugs, and then mixed for a short time. Afterwards the voltage was reapplied to separate the product from the unreacted substrate and the natural extract. The measured peak area of the product at 214 nm represents the enzyme activity. The electrophoretic conditions for the enzyme reaction and separation of substrate and product were optimized in this study. Under the optimal conditions, the Michaelis–Menten constant and the inhibitive mechanism of zanamivir were studied, which agreed with the literature data. Furthermore, the inhibitory ratios of enzymatic activity of depolymerized glycosaminoglycan and traditional Chinese drugs were determined. The EMMA method has superiority over traditional assay methods, in not only minimizing the false-positive results but also in simplifying the experimental procedure. Therefore, it could be employed to screen inhibitors from natural sources.

The transcription factors (TFs) plays an essential role in the cellular components to regulate gene expression, gene replication, gene repair, gene transcription and cell division. In this study, we proposed a simple, rapid and selective fluorescent method for monitoring TFs activity based on a exonuclease III (Exo III) cleavage-induced DNAzyme releasing strategy. A double-stranded DNA (dsDNA) which contains TFs binding site and 8–17 DNAzyme units was designed. With the addition of nuclear factor-kappa B p50 (NF-κB p50), the digestion of Exo III cannot proceed past the binding site, then the 8–17 DNAzyme substrate was successfully released and show its cyclic catalytic cleavage toward molecular beacon (MB) substrate, resulting in an evident fluorescence signal enhancement. With this currently developed Exo III cleavage reaction and DNAzyme-based platform, a detection limit of 6.2 pM could be achieved. Moreover, the inhibition effects of oridonin on NF-κB p50 have also been evaluated with satisfactory results. This developed Exo III cleavage-induced DNAzyme releasing strategy opens a promising avenue for monitoring activity and inhibition of nucleotide kinase, and should be also conveniently used for the sensor of other proteins or nucleic acid and may have widespread applications in biological process researches, drug discovery, and clinic diagnostics.

•The impurity in CMC is identified as diglycolic acid.•Pharmacological test indicated that diglycolic acid can cause BCT.•We established the refined method to eliminate the diglycolic acid in drug products.•Based on our findings, CFDA had issued a notice of companies to resume CMC products.Controlling and minimizing the adverse effects of drugs are the key issues in ensuring the safety of drug therapy. Carboxymethyl chitosan has been widely used as an anti-adhesion material. However, recently in China there have been several reported instances of conjunctival hyperemia associated with the use of carboxymethyl chitosan containing products. Through MS, FTIR, and GC analysis, an impurity, diglycolic acid, was discovered in carboxylmethyl chitosan products. Pharmacological tests further indicated diglycolic acid has antithrombogenicity properties and induces vasodilation, both of which can cause conjunctival hyperemia. Thus, through these tests it was ascertained that diglycolic acid was the culprit responsible for the adverse clinical effects. Next, emphasis shifted to trying to discover the mechanism responsible for generating the diglycolic acid. Under strong basic conditions, chloroacetic acid can generate glycolic acid, which, upon etherification, can become diglycolic acid. In order to avoid future adverse effects, we have established an HPLC method to detect and determine diglycolic acid in carboxymethyl chitosan products. This method is specific, accurate, and precise, and can be easily implemented into routine monitoring practice. Concurrently, a refined method has also been established in order to eliminate diglycolic acid from carboxymethyl chitosan.

•A method for preparing in-column tyrosinase microreactor based on LBL approach was developed for screening enzyme inhibitors.•Tyrosinase was immobilized on the surface of capillary via ionic binding technique with cationic polyelectrolyte HDB.•The method displays advantages including very low cost and reusable enzyme.•Inhibitors in natural extracts were screening by the present method.A method for creating an immobilized capillary tyrosinase (TRS) reactor based on a layer-by-layer (LBL) assembly for inhibitor screening is described. Tyrosinase was immobilized on the surface of fused-silica capillary via ionic binding technique with cationic polyelectrolyte hexadimethrine bromide (HDB). Then, HDB solution with the same plug length as the TRS was injected again into the capillary to cover the immobilized enzyme by forming HDB–TRS–HDB sandwich-like structure. Then, the substrate of l-tyrosine was introduced into the capillary and on-line enzyme inhibition study was performed by capillary electrophoresis (CE). The enzyme activity was determined by the quantification of peak area of the product of l-DOPA. Enzyme inhibition can be read out directly from the reduced peak area of the product in comparison with a reference electropherogram obtained in the absence of any inhibitor. The immobilized enzyme could withstand 25 consecutive assays by only losing 12% activity. A known TRS inhibitor, kojic acid was employed as a model compound for the validation of the inhibitor screening method. Finally, screening 19 natural extracts of traditional Chinese drugs was demonstrated. The results indicated that inhibition activity could be straightforwardly identified with the system.Figure optionsDownload full-size imageDownload as PowerPoint slide

Drug-protein binding is an important process in determining the activity of a pharmaceutical agent once it has entered the body. This paper developed an affinity capillary electrophoresis method to determine the binding constant between a bioactive sulfated polysaccharide 916 (916) and a potential protein, human serum albumin. This method is based on the principle that the changing analytes have different mobility shift in the zone electrophoresis. A fixed amount of protein was injected into a capillary filled with a background electrolyte containing the polysaccharide in varying concentrations. The effective mobility data of the protein were processed according to classical linearization treatments to obtain the binding constant of 916 to the HSA complex. The binding constant Ka of 916 to the human serum albumin achieved was 2.1 × 104.

A simple method for the determination of glucosamine and lactose in milk-based formulae by high-performance liquid chromatography (HPLC) with refractive index detector was developed and validated. Samples were cleaned up just by protein precipitation before HPLC analysis. Separation was achieved with a Shodex Asahipak NH2P-50 column. The method showed good linearity, sensitivity, precision and recovery, and proved very simple and rapid for routine analysis since separation was completely achieved at 10 min.