Preparation of solid lipid nanoparticles as drug carriers for levothyroxine sodium with in vitro drug delivery kinetic characterization.

The aim of this work was to produce and characterize solid lipid nanoparticles (SLN) containing levothyroxine sodium for oral administration, and to evaluate the kinetic release of these colloidal carriers. SLNs were prepared by microemulsion method. The particle size and zeta potential of levothyroxine sodium-loaded SLNs were determined to be around 153 nm,-43 mV (negatively charged), respectively by photon correlation spectroscopy. The levothyroxine entrapment efficiency was over 98%. Shape and surface morphology were determined by TEM and SEM. They revealed fairly spherical shape of nanoparticles.SLN formulation was stable over a period of 6 months. There were no significant changes in particle size, zeta potential and polydispersity index and entrapment efficiency, indicating that the developed SLNs were fairly stable.

Immobilisation of cobaltferritin onto gold electrode based on self-assembled monolayers.

The iron storage protein, ferritin, has a cavity of ∼7 nm in diameter in which iron is oxidised and stored as a hydrated oxide core. Electron transfer is known to be an important step in the sequestering of iron by cellular ferritin. The cavity was used as a nanocontainer to grow cobalt nanoparticles. The immobilisation of ferritin on the electrode surface is essential for various bioelectronic applications. A cobaltferritin-immobilised electrode based on self-assembled monolayer (SAM)-modified gold electrode was developed. The cobaltferritin-immobilised SAM-modified electrode was characterised by electrochemical and atomic force microscopy (AFM) techniques. The results indicated that cobaltferritin was selectively immobilised onto succinimidyl alkanedisulfide-modified Au electrode by the covalent interaction between cobaltferritin and the terminal functional groups of the SAMs. The cobaltferritin immobilised modified electrode showed a direct electron transfer reaction between cobaltferritin and the electrode. The electrochemically regulated uptake and release of cobalts for cobaltferritin immobilised on the SAMs were demonstrated. The results obtained in this study indicate that cobaltferritin has potential for a biomaterial in nanoscale synthesis for potential magnetic, catalytic and biomedical-sensing applications.

DNA-binding, DNA cleavage and cytotoxicity studies of two anthraquinone derivatives.

The interaction of native calf thymus DNA (CT-DNA) with two anthraquinones including quinizarin (1,4-dihydroxy anthraquinone) and danthron (1,8-dihydroxy anthraquinone) in a mixture of 0.04M Brittone-Robinson buffer and 50% of ethanol were studied at physiological pH by spectrofluorometric and cyclic voltammetry techniques. The former technique was used to calculate the binding constants of anthraquinones-DNA complexes at different temperatures. Thermodynamic study indicated that the reactions of both anthraquinone-DNA systems are predominantly entropically driven. Furthermore, the binding mechanisms on the reaction of the two anthraquinones with DNA and the effect of ionic strength on the fluorescence property of the system have also been investigated. The results of the experiments indicated that the binding modes of quinizarin and danthron with DNA were evaluated to be groove binding. Moreover, the cytotoxic activity of both compounds against human chronic myelogenous leukemia K562 cell line and DNA cleavage were investigated. The results indicated that these compounds slightly cleavage pUC18 plasmid DNA and showed minor antitumor activity against K562 (human chronic myeloid leukemia) cell line.

Assessment of antibody titers and immunity to Hepatitis B in children receiving chemotherapy.

BACKGROUND: There is a decrease in vaccine-specific antibody to certain vaccine-preventable diseases in children after chemotherapy, but the frequency of non-immune patients is not clear. In the present case-control study, was taken under investigation protection level to Hepatitis B infection in children 6 months after completing chemotherapy. MATERIALS AND METHODS: In this study 68 patients with cancer and 68 healthy children were enrolled. Patients were 1.5 -12 years old with completed standard chemotherapy at least for 6 months. All the patients and healthy children were negative for HBsAg and HBeAg and had received Hepatitis B vaccination. IgG antibody concentrations against Hepatitis B Virus (HBV) were determined in the patients receiving chemotrapy and healthy subjects serum by ELISA method. IgG antibody titer > 10 mIU/ml was considered as baseline protective titer for preventing HBV infection. RESULTS: Anti-HBs antibody titer in 19.12% of patients was less than 10 mIU/ml and 11.76% of the patients had borderline antibody titer (10-20 mIU/ml). In healthy subjects, 2.94% and 5.88% had antibody titer < 10 mIU/ml and 10-20 mIU/ml, respectively. According to statistical analysis, frequency of non immune subjects in children with cancer was significantly higher than those in healthy children (P-value=0.024). CONCLUSION: HBV vaccination post-intensive chemotherapy in the children with cancer is strongly recommended.

DNA-binding study of anthraquinone derivatives using chemometrics methods.

This work presents the interaction of two anthraquinones including quinizarin (1, 4-dihydroxyanthraquinone) and danthron (1, 8-dihydroxyanthraquinone) with CT-DNA in a mixture of Brittone-Robinson buffer (pH=7) with 50% of ethanol by UV-vis absorption, circular dichroism spectroscopic methods and viscosity measurements. The PCA and INDICES methods were used for predicting the number of light-absorbing components. Partially intercalative and hydrogen binding were found to be the dominant binding modes between anthraquinones and CT-DNA. The effect of ionic strength and pH on the behavior of the above system and also the interaction of anthraquinones with ds and ss-DNA were used to confirm the mentioned binding modes. The EQUISPEC software and nonlinear least-squares analysis were applied for binding constant determination.

DNA binding studies of PdCl2(LL) (LL = chelating diamine ligand: N,N-dimethyltrimethylenediamine) complex.

The interaction of native calf thymus DNA with the Pd(II) complex, PdCl2(LL) (LL = chelating diamine ligand: N,N-dimethyltrimethylenediamine), in 10 mM Hepes aqueous solutions at neutral pH has been monitored as a function of metal complex/DNA molar ratio by UV absorption spectrophotometry, circular dichroism (CD), viscosimetry, and fluorescence spectroscopy. The results support two modes of interaction. In particular, this complex showed absorption hypochromism and then hyperchromism, increase in melting temperature, and some structural changes in specific viscosity when bound to calf thymus DNA. The binding constant determined using absorption measurement is 2.69.10(3) M(-1). As evidenced by the increasing fluorescence of methylene blue-DNA solutions in the presence of increasing amounts of metal complex, PdCl(2)(LL) is able to displace the methylene blue intercalated into DNA, but not so completely, as indicated by partial intercalation. CD spectral changes in two steps and viscosity decrease confirm our conclusions.

Production and characterization of monoclonal antibody against human serum albumin.

Hybridomas secreting monoclonal antibodies (MAbs) producing stable, specific and high affinity against human serum albumin (HSA) have been established. The aim of the present study was the production of MAbs that will be potentially used in designing immunoassay methods especially immunochromatography assay kit for screening of microalbuminuria (MAU) in the early detection of diabetic and nondiabetic nephropathy. The hybridomas were obtained by fusion of spleen cells from immunized mice with mouse myeloma cell line (SP2). After limiting dilutions three clones producing antibodies were designed as EMRC1-3, which displayed different pattern of fine specificity for HSA and low cross reaction with other proteins as elucidated by inhibition enzyme-linked immunosorbent assay (ELISA). These clones were found to be of immunoglobulin G (IgG) class with k light chain. Subclass determination showed that all three MAbs secreted IgG1 type of antibody. The results of affinity purification for the two selected clones (EMRC1 and EMRC3) displayed high affinity with no cross reactivity with any of the related protein molecules. The stable hybridomas secreting anti-HSA were expanded in 50-mL flasks for large-scale production of the required antibodies. The standard curves were constructed with a sensitivity of 10 pg per well covering up to 100 ng per well. The high binding activity to HSA antigen and having no cross reactivity with other related molecules illustrated the potential application of these antibodies as an immunodiagnostic reagent in designing an immunochromatography assay kit for screening of MAU in diabetic and nondiabetic patients.

DNA interaction with Al-N,N'-bis(salicylidene)2,2'-phenylendiamine complex.

The Al(III) complex, [Al(salophen)2H2O]NO3, was synthesized and characterized by spectroscopic (NMR and FT-IR) techniques. Then the binding of Schiff base complex of [Al(salophen)]+ type, where salophen denotes N,N'-bis(salicylidene) 2,2-phenylendiamine to calf thymus DNA, has been investigated by spectrophotometric, circular dichroism, spectrofluorometric, melting temperature and viscosimetric techniques. This Al(III) complex showed absorption hyperchromism in the range of 310-390 nm, increase in melting temperature, some structural changes in specific viscosity, when bound to calf thymus DNA. The binding constant has been determined using absorption measurement and found to be 1.82 x 10(3)M(-1) and 1.31 x 10(3)M(-1) in HEPES and Tris-HCl buffers, respectively. Also the fluorescence spectral characteristics and interaction of Al-salophen complex with DNA have been studied. Al-salophen bound to DNA showed a marked increase in the fluorescence intensity along with a bathochromic shift (5 nm). The intersection point of the binding isotherm indicated a binding site size of 12 bp per bound complex molecule in both HEPES and Tris-HCl buffers. The experimental results showed that the Al-salophen complex bound to DNA by non-intercalative mode and major groove binding was the preferred mode of interaction.

Preparation and characterization of a monoclonal antibody against mannoprotein of Candida albicans.

BALB/c mice were immunized via injection with whole cell of Candida albicans serotype A. The spleens were fused with myeloma cells of SP2/0 origin. A mannoprotein-reactive monoclonal antibody (MAb) was selected and characterized by ELISA technique. This MAb reacted with strains of Candida such as C. albicans, C. tropicalis, and C. albicans of the Persian Type Culture Collection (PTCC). However, our antibody did not react with other Candida species such as C. parapsilosis, C. glabrata, C. stellatoidae, C. lusitania, C. krusei, and S. cervisiae. These antibodies also did not recognize extracts of other fungal species such as Aspergillus fumigatus and Aspergillus flavus, and bacterial strains such as Staphylococcus aureus and Pseudomonas aeruginosa. Polyclonal antibody produced in this study could not differentiate the above species and was reactive towards all fungal species mentioned above except bacterial strains of S. aureus and P. aeruginosa. Western blot analysis of ligand affinity-purified mannoproteins of C. albicans wall protein using this MAb showed reactivity toward a single protein band in the region of 55-65 kDa molecular weight. The same antibody, when examined with unpurified C. albicans extract, reacted with a broad band in the region of 55-105 kDa, which we concluded was due to a possible different glycosylation pattern of mannoprotein in crude extract in which the higher molecular weight protein was eliminated by ligand-binding affinity purification.

Production and characterization of a new antibody specific for the mutant EGF receptor, EGFRvIII, in Camelus bactrianus.

EGFRvIII is the type III deletion mutant form of the epidermal growth factor receptor (EGFR) with transforming activity. This tumor-specific antigen is ligand independent, contains a constitutively active tyrosine kinase domain and has been shown to be present in a number of human malignancies. In this study, we report the production and characterization of camel antibodies that are directed against the external domain of the EGFRvIII. Antibodies developed in camels are smaller (i.e. IgG2 and IgG3 subclasses lack light chains) than any other conventional mammalian antibodies. This property of camel antibodies makes them ideal tools for basic research and other applications such as tumor imaging and cancer therapy. In the present study, camel antibodies were generated by immunization of camelids (Camelus bactrianus and Camelus dromedarius) with a synthetic 14-amino acid peptide corresponding to the mutated sequence of the EGFR, tissue homogenates of several patients with human glioblastoma, medulloblastoma and aggressive breast carcinoma, as well as EGFR-expressing cell lines. Three subclasses of camel IgG [conventional (IgG1, 160 kD) and heavy chain-only antibodies (IgG2 and IgG3, 90 kD)] were separated by their different binding properties to protein A and protein G affinity columns. The anti-EGFRvIII peptide antibodies from immunized camels were purified further using the EGFRvIII synthetic peptide affinity column. The purified anti-EGFRvIII peptide camel antibodies selectively bound to the EGFRvIII peptide and affinity-purified EGFRvIII from malignant tissues and detected a protein band of 140 kD from malignant tissues by Western blot. Affinity analysis showed that the antibodies from C. bactrianus and C. dromedarius reacted with peptide and antigen purified from a small cell lung cancer ascitic fluid with affinities of 2 x 10(8) and 5 x 10(7)M(-1) to the same extent, respectively. Since the functional antigen-binding domain of the anti-EGFRvIII antibodies in camels is much simpler and located only on the heavy chains of proteins, we are currently developing recombinant and smaller versions of the variable domain of these naturally occurring heavy-chain antibodies (V(HH)) for use in tumor imaging and cancer therapy.

Production of monoclonal antibody, PR81, recognizing the tandem repeat region of MUC1 mucin.

A monoclonal antibody (MAb) was generated by immunizing BALB/c mice with homogenized breast cancerous tissues. This antibody (PR81) was found to be of IgG(1) class and subclass, containing kappa light chain. PR81 reacted with either the membrane extracts of several breast cancerous tissues or the cell surface of some MUC1 positive cell lines (MCF-7, BT-20 and T-47D) tested by enzyme immunoassay and for MCF-7 by immunofluorescence method. PR81 also reacted with two synthetic 27 and 16-amino acid peptides, TSA-P1-24 and A-P1-15, respectively, which included the core tandem repeat sequence of MUC1. However, this antibody did not react with a synthetic 14 amino acid peptide that has no similarity with tandem repeat found in MUC1. The generated antibody had good and similar affinities (2.19 x 10(8) M(-1)) toward TSA-P1-24 and A-P1-15, which are mainly shared in the hydrophilic sequence of PDTRPAP. Through Western blot analysis of homogenized breast tissues, PR81 recognized only a major band of 250 kDa. This band is stronger in malignant tissue than benign and normal tissues.

Preparation and characterization of monoclonal antibody against digoxin.

Mouse-mouse hybridoma cell lines producing stable, highly specific and with good affinity monoclonal antibody (MAb) against the cardiac glycoside digoxin were established. Balb/c mice were immunized via injection of digoxin-3'-bovine serum albumin (BSA). The spleens of which were fused with myeloma cells of SP2/0 origin. Three clones designated as BBA, MBE, and BMG producing good antibodies displayed different patterns of fine specificity for digoxin and low cross-reaction with several digoxin analogues as elucidated by inhibition enzyme-linked immunosorbant assay (ELISA). All three MAbs were of the same class and subclass (IgG(1)). Affinity purification was performed for the selected clone BBA displaying the highest affinity and nearly no cross-reactivity with any of the structurally related molecules. Ultrafiltered concentrated hybrid cell supernatant was also purified by polyethylene glycol (PEG) 6000 precipitation for large-scale preparation and coated onto the wells of microtiter plates. The standard curve was constructed with a sensitivity of 10 pg/well covering up to 10 ng/well.

Spectrophotometric study of the alkali metal-murexide complexes in some non-aqueous solutions.

The complexes of murexide with alkali-metal cations have been studied spectrophotometrically in methanol, dimethylformamide and dimethylsulphoxide media at 25 degrees . The stoichiometry of the complexes was found to be 1:1. The formation constants of the complexes were determined, and found to decrease in the order Na(+) > K(+) > Rb(+) approximately Li(+) for all solvents studied. The complex formation constants varied inversely with the Gutmann donicity of the solvents.