The grafting and release behavior of doxorubincin from Fe(3)O(4)@SiO(2) core-shell structure nanoparticles via an acid cleaving amide bond: the potential for magnetic targeting drug delivery.

Fe(3)O(4)@SiO(2) core-shell structure nanoparticles were first prepared and characterized by TEM, FTIR, XPS and XRD. Subsequently the widely used anticancer agent doxorubincin (DOX) was successfully grafted to the surface of the core-shell nanoparticles via an amide bond with the aid of a spacer arm we synthesized. The spacer arm met two needs: one end can couple to the core-shell nanoparticles' surface while the other end was the active -COOH group, which can react with the -NH(2) group of DOX molecules. The synthesized spacer arm and the conjugation of the drug with nanoparticles through amidation were confirmed by FTIR. The DOX-loading efficiency determined by UV-vis spectrometer was 86.5%. Drug release experiments displayed a pH-dependent behavior that DOX was cleaved from the nanoparticles easily under low pH conditions in the presence of protease and that most of the conjugated doxorubincin were released within the first 12 h. The prepared DOX-grafted Fe(3)O(4)@SiO(2) core-shell structure nanoparticles showed a superparamagnetic property with a saturation magnetization value of 49.3 emu g(-1), indicating a great potential application in the treatment of cancer using magnetic targeting drug-delivery technology.

Human catalytic antibodies with glutathione peroxidase activity.

In order to generate catalytic antibodies with glutathione peroxidase (GPx) activity, we prepared GSH-S-DNP butyl ester and GSH-S-DNP benzyl ester as the haptens. Two ScFvs that bound specifically to the haptens were selected from the human phage-displayed antibody library. The two ScFv genes were highly homologous, consisting of 786 bps and belonging to the same VH family-DP25. In the premise of maintaining the amino acid sequence, mutated plasmids were constructed by use of the mutated primers in PCR, and they were over-expressed in E. coli. After the active site serine was converted into selenocysteine with the chemical modifying method, we obtained two human catalytic antibodies with GPx activity of 72.2U/micromol and 28.8U/micromol, respectively. With the aid of computer mimicking, it can be assumed that the antibodies can form dimers and the mutated selenocysteine residue is located in the binding site. Furthermore, the same Ping-Pong mechanism as the natural GPx was observed when the kinetic behavior of the antibody with the higher activity was studied.

[An abzyme to catalytize the deiodination of thyroxine].

AIM: To mimic an important family of selenoenzymes in organism-thyroxine (T4) deiodinases and prepare a selenium-containing abzyme catalyzing deiodination of T4. METHODS: A anti-T4 monoclonal antibody was generated by hybridoma methodology and converted into a selenium-containing abzyme by the method of chemical modification. The catalytic activity of the enzyme was measured by RIA method. RESULTS: The abzyme displayed a marked activity of catalyzing deiodination of T4 and a higher specificity to the substrate T4 than that of natural enzyme, and the double reciprocal plots of the initial rates of T3 formation vs. T4 concentration yielded a family of parallel lines. The catalytic activity could be sensitively inhibited by 6-propyl-2-thiouracil (PTU), a competitive inhibitor for dithiothreitol (DTT). CONCLUSION: An abzyme with the diodination activity was first prepared and the reaction mechanism of the enzyme was bisubstrate ping-pong one.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of phospholipase A2 and fibrinolytic enzyme, two enzymes obtained from Chinese Agkistrodon blomhoffii Ussurensis venom.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to analyze two enzymes, phospholipase A2 and fibrinolytic enzyme isolated from Chinese Agkistrodon blomhoffii Ussurensis venom. Using sinapinic acid as the matrix, positive ion mass spectra of the enzymes were obtained. In addition to the dominant protein [M + H]+ ions, multimeric and multiply charged ions were also observed in the mass spectra. The higher the concentration of the enzymes, the more multiply charged polymer and multimeric ions were detected. Our results indicate that MALDI-TOFMS can provide a rapid and accurate method for molecular weight determination of snake venom enzymes. Mass accuracies of 0.1 and 0.3% were achieved by analysis of highly dialyzed phospholipase A2 and fibrinolytic enzyme, and these results are much better than those obtained using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. MALDI-TOFMS thus provides a reliable method to determine the purity and molecular weight of these enzymes, which are of potential use as therapeutants.

Effects of La3+ on lipid fluidity and structural transitions in human erythrocyte membranes.

The effects of La3+ on the structure and function of human erythrocyte membranes were investigated by fluorescence polarization, spin-labeled electron spin resonance (ESR) and differential scanning calorimetry (DSC). The results showed that increasing concentrations of La3+ inhibited (Na(+) + K+)-ATPase and Mg(2+)-ATPase activities. La3+ lowered the lipid fluidity of erythrocyte membranes and induced structural transitions in erythrocyte membranes.

Induction of a dose-related increase in sulfobromophthalein uptake velocity in freshly isolated rat hepatocytes by phenobarbital.

To determine whether phenobarbital affects hepatocellular bilirubin/sulfobromophthalein uptake mechanism, we administered it to male Sprague-Dawley rats, body weight 175 +/- 25 gm, at doses of 1 to 75 mg/kg body wt/day for 7 days. Control rats were given an equivalent volume of physiological saline solution. On day 8, hepatocytes were isolated by means of collagenase perfusion, suspended in Hanks' solution without albumin and incubated with high specific activity (3 Ci/mmol) [35S]sulfobromophthalein, which was synthesized in our laboratory and purified by means of a new reverse-phase high-pressure liquid chromatography procedure. The initial uptake rate of sulfobromophthalein was determined at sulfobromophthalein concentrations of 1 to 50 mumol/L with a rapid filtration technique. The maximum uptake velocity and Michaelis constant for sulfobromophthalein uptake at each phenobarbital dose were determined by means of a computer analysis. In control studies, maximum uptake and Michaelis constant were 48.0 +/- 16.7 (mean +/- S.D.) pmol/50,000 cells/min and 22 +/- 4 mumol/L, respectively. Maximum uptake velocity increased linearly with the log of the phenobarbital dose (r = 0.98, p < 0.01), the increase achieving statistical significance at a dose of 3 mg/kg/day. Michaelis constant, however, was essentially unchanged at phenobarbital doses of 50 mg/kg/day or less. The maximal observed increase in maximum uptake velocity of sulfobromophthalein (to 619% of control values) was appreciably greater than the maximal increase in UDP-glucuronyltransferase activity (200% of control) or immunoreactive ligandin concentrations (260% of control) seen in earlier studies, suggesting a direct effect on the plasma membrane transport mechanism.

Effect of lanthanide ions on the phase behavior of dipalmitoylphosphatidylcholine multilamellar liposomes.

The effect of lanthanide ions (Ln3+) and their coordination compounds of diethylenetriamine pentaacetic acid (DTPA) on the phase behavior of dipalmitoylphosphatidylcholine (DPPC) multi-lamellar liposomes has been studied by differential scanning calorimetry (DSC), Raman spectroscopy, and freeze-fracture electron microscopic techniques. The displacement of Ca2+ binding on DPPC liposomes by lanthanide ions was also studied. The results show that the binding degree of four kinds of chloride salts with DPPC liposomes is: YbCl3 > GdCl3 > LaCl3 > CaCl2. Lanthanide ions increase the phase transition temperature of DPPC liposomes and decrease the membrane fluidity. Freeze-fracture electron microscopic results show that La3+ enhances the order of DPPC membrane. The effect of coordination compounds of lanthanides with DTPA on the phase behavior of DPPC liposomes is smaller than that of their chlorides. La3+, Gd3+, and Yb3+, can displace Ca2+ binding on DPPC liposomes, but there coordination compounds of DTPA can hardly displace Ca2+. Raman spectroscopic results show that a very slight effect in lateral packing order of DPPC liposomes was observed at various concentrations of lanthanides.

Direct observation of molecular images of lanthanide phthalocyanines: III. Structural defects.

The crystal imperfections in thin films of lanthanide phthalocyanines (LnPc2H, Ln = Nd, Tb, Er, Tm, Yb, and Lu) grown expitaxially on KCl have been observed by molecular imaging. Grain and twin boundaries, stacking faults, point defects, vacancies, mosaic structures, and sometimes even some amorphous islands exist in the well-crystallized specimens. Combined with the results reported earlier, the packing characteristics of planar LnPc2H molecules can be well understood.