Preparation and In Vitro Evaluation of a MRI Contrast Agent Based on Aptamer-Modified Gadolinium-Loaded Liposomes for Tumor Targeting.

The aim of this study was to prepare aptamer-modified liposomes loaded with gadolinium (Gd) to enhance the effective diagnosis for tumor by MRI. A modified GBI-10 (GBI-10m) was used to prepare targeted liposomes (GmLs). Liposomes with GBI-10 aptamer (GLs) and without aptamer (non-targeted liposomes (NLs)) were also prepared as controls. The particle size and zeta potential of GmLs, GLs, and NLs were all assayed. A clinical 3.0 T MR scanner was employed to assess the imaging efficiency and measure the longitudinal relaxivity (r 1) of the above liposomes. Confocal laser scanning microscopy and flow cytometry were used to analyze and compare the targeting effects of GmLs, GLs, and NLs to MDA-MB-435s cells at 37°C. The particle size of the prepared liposomes was scattered at 100-200 nm, and their values of r 1 were ∼4 mM-1 s-1. The images of confocal laser scanning microscopy showed that GmLs in the cytoplasm were significantly more than GLs and both GmLs and GLs were more than NLs. The fluorescence intensity of GmLs was increased by about two times than that of GLs and three times than that of NLs by flow cytometry. Therefore, the GmLs in this initial study were suggested to be a potential MRI contrast agent at 37°C for diagnosing tumors with the protein of tenascin-C over-expressed.

Characterization of the molecular properties and allergenicity (IgE-binding capacity) of ß-lactoglobulin by heat treatment using asymmetric-flow field-flow fractionation and ultra-performance liquid chromatography quadrupole time of flight mass chromatography.

In this study, the heat product (90 °C, 10 min) of ß-lactoglobulin (ß-LG) was analyzed by asymmetric-flow field-flow fractionation (AF4) to observe the effect of heat treatment. The changes in molar mass (M) and molar size induced by heat treatment were characterized by AF4, and changes in molar shape were observed by transmission electron microscopy (TEM). The results showed that ß-LG dissociated and aggregated into four fractions with different M values, sizes, and shapes after heat treatment. The vast aggregations with the highest allergenicity (IgE-binding capacity) might enhance the allergenicity of ß-LG. However, the number of characterized epitope peptides was decreased due to heat treatment. The above results provide some references for related studies of ß-LG and its allergenicity. Further separation and characterization of the high-allergenicity fractions and peptides will help to eliminate allergens in dairy products and reduce the occurrence of allergic reactions.

Combination of magnetic beads extraction and ultraperformance liquid chromatography tandem mass spectrometry detection for the clinical diagnosis of allergies.

The total amount of immunoglobulin E (IgE) in human serum is an important parameter in diagnosing allergies. To reduce the false diagnosis of allergies and better assist in therapy, clinical studies can be performed to obtain accurate and reliable measurements of IgE. A magnetic beads (MBs)-based ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for total IgE measurement and the diagnosis of food allergies in serum was developed in this study. First, IgE was extracted by MBs coupled with anti-IgE antibody from serum. The extracted IgE was quantified by a specific signal peptide after digestion. A spiked linear IgE concentration ranging from 400 to 5000 ng mL-1 was used for quantification. The limits of detection and quantification were 400 ng mL-1 and 800 ng mL-1, respectively, for the developed method. Additionally, the combined capacity of the extracted IgE with different allergic proteins was evaluated by a binding experiment in vitro. The combining capacity of IgE with different allergens was used to speculate the kind of allergens that induce allergies in patients. Overall, a new method was developed that could be used to quantify the amount of IgE and simultaneously diagnose which allergen causes an allergic reaction, and this method may provide a powerful new tool in the clinical detection of allergies. Moreover, the developed method was applied to analyze IgE in four samples of patient serum and four serum samples from healthy individuals.

[Asymmetrical flow field flow fractionation combined with ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry used to screen allergen protein epitopes].

Asymmetrical flow field flow fractionation (AF4) combined with ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used to screen allergen protein epitopes. The selected allergen protein (tropomyosin, TM) was enzymatically digested into peptide segments and analyzed via UPLC-QTOF-MS to establish a protein-specific peptide database. The peptide segments were incubated with immunoglobulin E (IgE) for 30 min. During the incubation procedure, the specific peptide segments (with the antigen epitope) combine with IgE while the other peptide segments remain in solution. After incubation, the solution was injected into the AF4 device. The combined peptide segments flowed out of the outlet along with IgE, and the other peptide segments flowed into the waste liquid. The components of outlet were then collected, analyzed by UPLC-QTOF-MS, and the results matched with the spectra of the protein peptides. Eventually the specific peptide segments were identified to detect the antigen epitopes. This study extends the application of AF4 with a preliminary exploration of the detection of an allergen protein epitope, providing a novel research strategy for the screening of allergen epitopes.

[Impact of carrier flow composition on membrane adsorption and aggregation of ovalbumin in asymmetrical flow field-flow fractionation].

Asymmetrical flow field-flow fractionation (AF4) is a kind of moderate separation technology for the analysis of macromolecules, including proteins with a wide range of sizes. In the separation channel, the membrane adsorption and aggregation of proteins affected by the carrier fluid (CF) composition lead to changes in analyte recovery and size distribution, thereby restricting the application of AF4 to biomolecules. Different pH levels (6.2, 7.4, 8.2), several types of cations (Na+, K+, Mg2+) and various ion strengths (0-0.1 mol/L)were studied to demonstrate the influence of carrier fluid composition on the membrane adsorption and aggregation of proteins. The results revealed the following:a) higher ion strength of CF resulted in a greater degree of membrane adsorption and aggregation; b) the zeta potential, determined by the pI of the protein and the pH of the CF, influenced the adsorption and aggregation; c) divalent cations (Mg2+) caused serious adsorption and aggregation. The experimental results can help us achieve better recovery and mitigate aggregate formation by using the optimal CF components in future AF4 studies. Moreover, the findings indicate that AF4 would find extensive application in protein biochemistry assays.

[Present situation and development trends of asymmetrical flow field-flow fractionation].

Field-flow fractionation (FFF) is a kind of mature separation technologies in the field of bioanalysis, feasible of separating analytes with the differences of certain physical and chemical properties by the combination effects of two orthogonal force fields (flow field and external force field). Asymmetrical flow field-flow fractionation (AF4) is a vital subvariant of FFF, which applying a vertical flow field as the second dimension force field. The separation in AF4 opening channel is carried out by any composition carrier fluid, universally and effectively used in separation of bioparticles and biopolymers due to the non-invasivity feature. Herein, bio-analytes are held in bio-friendly environment and easily sterilized without using degrading carrier fluid which is conducive to maintain natural conformation. In this review, FFF and AF4 principles are briefly described, and some classical and emerging applications and developments in the bioanalytical fields are concisely introduced and tabled. Also, special focus is given to the hyphenation of AF4 with highly specific, sensitive detection technologies.

[Effects of carrier liquid and flow rate on the separation in gravitational field-flow fractionation].

Gravitational field-flow fractionation is the simplest field-flow fractionation technique in terms of principle and operation. The earth' s gravity is its external field. Different sized particles are injected into a thin channel and carried by carrier fluid. The different velocities of the carrier liquid in different places results in a size-based separation. A gravitational field-flow fractionation (GrFFF) instrument was designed and constructed. Two kinds of polystyrene (PS) particles with different sizes (20 µm and 6 µm) were chosen as model particles. In this work, the separation of the sample was achieved by changing the concentration of NaN3, the percentage of mixed surfactant in the carrier liquid and the flow rate of carrier liquid. Six levels were set for each factor. The effects of these three factors on the retention ratio (R) and plate height (H) of the PS particles were investigated. It was found that R increased and H decreased with increasing particle size. On the other hand, the R and H increased with increasing flow rate. The R and H also increased with increasing NaN3 concentration. The reason was that the electrostatic repulsive force between the particles and the glass channel wall increased. The force allowed the samples approach closer to the channel wall. The results showed that the resolution and retention time can be improved by adjusting the experimental conditions. These results can provide important values to the further applications of GrFFF technique.

Effects of comprehensive function of factors on retention behavior of microparticles in gravitational field-flow fractionation.

Gravitational field-flow fractionation (GrFFF) is a useful technique for separation and characterization for micrometer-sized particles. Elution behavior of micrometer-sized particles in GrFFF was researched in this study. Particles in GrFFF channel are subject to hydrodynamic lift forces (HLF), fluid inertial forces and gravity, which drive them to different velocities by carrier flow, resulting in a size-based separation. Effects of ionic strength, flow rate and viscosity as well as methanol were investigated using polystyrene latex beads as model particles. This study is devoted to experimental verification of the effect of every factor and their comprehensive function. All experiments were performed to show isolated influence of every variable factor. The orthogonal design test was used to evaluate various factors comprehensively. Results suggested that retention ratio of particles increases with increasing flow rate or the viscosity of carrier liquid by adjusting external forces acting on particles. In addition, retention ratio increases as ionic strength decreases because of decreased electrostatic repulsion between particles and channel accumulation wall. As far as methanol, there is no general trend due to the change of both density and viscosity. On the basis of orthogonal design test it was found that viscosity of carrier liquid plays a significant role in determining resolution of micrometer-sized particles in GrFFF.

[Determination of six anticoccidials in chicken using QuEChERS combined with ultra high liquid chromatography-high resolution mass spectrometry].

An ultra high liquid chromatography-Q Exactive orbitrap mass spectrometry multi-residue method has been developed for the determination of six anticoccidials residues (dinitlmide, nicarbazin, diclazuril, toltrazuril, monensin and salinomycin) in chicken tissue. Sample preparation was based on QuEChERS method, using 1% (v/v) trichloroacetic acid/acetonitrile aqueous solution (3:7, v/v) as the extraction solvent and salting-out with sodium chloride followed by clean-up with 50 mg/mL primary secondary amine (PSA) +50 mg/mL neutral alumina (Alumina-N) dispersive solid phase extraction (DSPE). The separation of the compounds in liquid chromatography was carried out using a Waters Acquity UPLC BEH C8 column (100 mm x 2.1 mm, 1.7 µm) with mobile phases consisting of methanol-5 mmol/L ammonium acetate aqueous solution in gradient elution. The Q Exactive orbitrap mass spectrometric detection was carried out with positive and negative electrospray ionization simultaneously. The results showed the linear ranges of the six target compounds were as follows: dinitolmide, 1.0-30.0 µg/L; nicarbazin, 0.2-6.0 µg/L; diclazuril and toltrazuril, 2.0-60.0 [µg/L; monensin and salinomycin, 4.0-120.0 µg/L. The external standard method was used for quantification. The spiked recoveries at three levels for the six anticoccidials ranged from 67.7% to 126.8%. The relative standard deviations (RSDs) were ≤ 10.4%. The limits of quantification (LOQs) were as follows: dinitolmide, 2.50 µg/kg; nicarbazin, 0.50 µg/kg; diclazuril and toltrazuril, 5.00 µg/kg; monensin and salinomycin, 20.00 µg/kg. The developed method is easy of operation and of high sensitivity. It can meet the requirements of daily inspection.

In vitro study of novel gadolinium-loaded liposomes guided by GBI-10 aptamer for promising tumor targeting and tumor diagnosis by magnetic resonance imaging.

Novel gadolinium-loaded liposomes guided by GBI-10 aptamer were developed and evaluated in vitro to enhance magnetic resonance imaging (MRI) diagnosis of tumor. Nontargeted gadolinium-loaded liposomes were achieved by incorporating amphipathic material, Gd (III) [N,N-bis-stearylamidomethyl-N'-amidomethyl] diethylenetriamine tetraacetic acid, into the liposome membrane using lipid film hydration method. GBI-10, as the targeting ligand, was then conjugated onto the liposome surface to get GBI-10-targeted gadolinium-loaded liposomes (GTLs). Both nontargeted gadolinium-loaded liposomes and GTLs displayed good dispersion stability, optimal size, and zeta potential for tumor targeting, as well as favorable imaging properties with enhanced relaxivity compared with a commercial MRI contrast agent (CA), gadopentetate dimeglumine. The use of GBI-10 aptamer in this liposomal system was intended to result in increased accumulation of gadolinium at the periphery of C6 glioma cells, where the targeting extracellular matrix protein tenascin-C is overexpressed. Increased cellular binding of GTLs to C6 cells was confirmed by confocal microscopy, flow cytometry, and MRI, demonstrating the promise of this novel delivery system as a carrier of MRI contrast agent for the diagnosis of tumor. These studies provide a new strategy furthering the development of nanomedicine for both diagnosis and therapy of tumor.

Electrochromatographic properties of synthetic poly(styrene-divinylbenzene) encapsulated packing material and sulphonic cation-exchange phase based on the former.

OBJECTIVE: To develop new kinds of stationary phases suitable for applications in capillary electrochromatography (CEC), as well as in LC, which can be tailored to realize selective separations of solutes difficult to separate with conventional stationary phases. METHODS: Purchased spherical silica particles (5 microm) were refluxed in anhydrous toluene with vinyl-triethoxysilane for 18 h, to modify the surface with vinylsilyl groups. The silylated silica particles were subsequently stirred in an n-henanol-acetonitril mixture (1:4, volume fraction) for 18 h. Styrene and divinylbenzene in the mixture co-polymerized to form a crosslinked encapsulation layer on the silica surface, and bonded on the surface vinyl groups at the same time, both reactions were initiated by azo-iso-butyronitrile. The poly(Styrene-divinylbenzene) encapsulated silica(PS-DES) produced can be used as a non-conventional stationary phase for CEC itself. By sulphonation of the PS-DES phase with chlorosulphonic acid, strong cation-exchange stationary phase of sulphonic type was obtained. RESULTS: The manufactured PS-DES as well as the sulphonated phases were respectively packed into capillaries,and the columns thus prepared were tested for their chromatographic characteristics. It was found that the PS-DES phase showed reversed-phase characteristics. Due to the phenyl groups in the encapsulated polymer, it introduced pi-pi electronic interaction between the solutes molecules and stationary phase during the chromatographic separation process, therefore it showed unique selectivity on separating aromatic compounds, also polar- as well as some alkaline drugs was analyzed on the column packed with the phase. It was demonstrated that the sulphonated phase could be used to prepare columns for the separation of alkaline drugs, symmetric peaks were obtained for them and base-line separation was realized. CONCLUSION: It is possible that these stationary phases prepared can be used for solving the analytical problems in which non-conventional selectivity are needed ( the analyses of alkaline drugs are examples) In some cases, the analysts can realize the anticipated separation results based on the different separation mechanism from that of the conventional stationary phases.

Mechanism underlying carbon tetrachloride-inhibited protein synthesis in liver.

AIM: To study the mechanism underlying carbon tetrachloride (CCl(4)) -induced alterations of protein synthesis in liver. METHODS: Male Sprague-Dawley rats were given CCl(4) (1 mL/100 g body weight) and (3)H-leucine incorporation. Malondialdehyde (MDA) level in the liver, in vitro response of hepatocyte nuclei nucleotide triphosphatase (NTPase) to free radicals, and nuclear export of total mRNA with 3'-poly A(+) were measured respectively. Survival response of HepG2 cells to CCl(4) treatment was assessed by methyl thiazolyl tetrazolium. Km and Vmax values of nuclear envelope NTPase activity in liver of rats treated with CCl(4) were assayed by a double-reciprocal plot. RESULTS: The protein synthesis was inhibited while the MDA level was significantly increased in liver of rats treated with CCl(4). In addition, CCl(4) decreased the NTPase binding capacity of nuclear envelope (Km value) in cultured HepG2 cells. Moreover, in vitro ferrous radicals from Fenton's system suppressed the NTPase activity of liver nuclear envelope in a dose-dependent manner. Down-regulation of the nuclear envelope NTPase activity indicated a lower energy provision for nucleocytoplasmic transport of mRNA molecules, an evidence in CCl(4)-treated HepG2 cells correspondingly supported by the nuclear sequestration of poly (A)(+) mRNA molecules in morphological hybridization research. CONCLUSION: Inhibition of mRNA transport, suggestive of decreased NTPase activity of the nuclear envelope, may be involved in carbon tetrachloride-inhibited protein synthesis in liver.

Immobilization of phospholipid-avidin on fused-silica capillaries for chiral separation in open-tubular capillary electrochromatography.

Phospholipid-coated fused-silica capillaries with immobilized avidin were applied in the chiral separation of D,L-tryptophan, D,L-PTH-serine, and D,L-PTH-threonine at pH 7.4 by open-tubular CEC. Liposomes prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap biotinyl), or 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Biotinyl) with different amounts of phosphatidylserine were assessed as phospholipid coating materials. The stability of the coating and the success of the coating procedure were evaluated in terms of the repeatability of the enantiomer migration times and the resolution of enantiomers. The coating procedure itself significantly affected the migration times and resolution of the enantiomers. Reliable chiral separations with high separation efficiencies were achieved through careful choice of the coating method.

Chromatographic behavior of packing materials for capillary electrochromatography with a coating of different immobilized polysiloxanes.

Octadecyl silyl silica (ODS) phase coated with immobilized polysiloxanes (OV1701, SE-54, SE-30) were synthesized, their characteristics as capillary electrochromatography (CEC) column packing materials were studied. It was found that, although the polysiloxane coatings were different in polarity, the resulting packing materials showed the highest efficiencies when the respective coating ratios (polysiloxane:ODS, w/w) were all 20-30%. As expected, packing materials coated with different polysiloxanes resulted in different selectivity on solute pairs. Separations on these stationary phases were studied with different factors such as pH values and acetonitrile contents of the mobile phases. It was found that all these kind of stationary phases could resist basic mobile phase with a pH value as high as 11.6. Tests were made to analyze polar, basic drugs with CEC using the stationary phases.