Mutual effects of protein corona formation on CdTe quantum dots.

Future biomedical applications of nanoparticles will encounter these particles with patients' serum which might affect the properties and stability of quantum dots and serum proteins at the desired site of action. Therefore, it is essential to clarify the patient-specific serum components, serve as major interaction partners, the spatial distribution of these, and consequently the time-dependent effects of nanoparticle-protein interaction. Here, a biochemical and structural study was performed on the protein corona formation and the corresponding interaction of different sizes of CdTe QDs with human serum proteins to determine if the mutual effects on optical properties by using electrophoresis, chemiluminescence, and fluorescence spectroscopy. The results revealed that interaction with human serum significantly enhanced the stability and photoluminescence of quantum dots. Structural studies of HSA-coated CdTe QDs also showed that corona formation has no adverse effects on protein structure, and the reduction in fluorescence emissions of HSA is due to the direct quenching of aromatics residues by the quantum dot. Improving nanoparticle properties, as well as the lack of structural changes in HSA, can be very useful in biomedical applications and in vivo studies where stability is important.

Targeted anticancer prodrug therapy using dextran mediated enzyme-antibody conjugate and ß-cyclodextrin-curcumin inclusion complex.

A targeted and controlled drug delivery system based on ß-cyclodextrin (ß-CD) for encapsulation and controlled release of hydrophobic drugs in the presence of maltogenic amylase (MAase), as a cyclodextrin-hydrolyzing enzyme, and trastuzumab antibody has been developed. In this study, the inclusion complex of curcumin (CUR), as a model anticancer compound, with ß-CD was prepared and we constructed an antibody-enzyme bioconjugate (dextran mediated MAase-Trastuzumab bioconjugate) for controlled and targeted release of CUR at HER2 positive cancer cells (including SKBR3 and BT474). Immunocytochemistry analysis indicated that the MAase-Trastuzumab bioconjugate had significant binding affinities to HER2 positive cancer cells and demonstrated high enzyme activity to degrade ß-CD in order to rapid release of CUR on targeted cell surface. Fluorescence microscopy images and cytotoxicity studies represent significantly greater cellular uptake and anti-proliferative effects of CUR by ß-CD-CUR/MAase-Trastuzumab bioconjugate compared to free CUR and ß-CD-CUR in presence and absence of MAase in HER2 positive cells. The results from flow cytometric assay suggest that the ß-CD-CUR/MAase-Trastuzumab conjugate exhibited higher cytotoxic and apoptotic effects on cancer cells compared to other formulation. We demonstrate that this formulation has a potential application for targeted and controlled release of drugs in cancer therapy with increased therapeutic efficiency.

Enhanced sensitivity of VEGF detection using catalase-mediated chemiluminescence immunoassay based on CdTe QD/H2O2 system.

BACKGROUND: Since vascular endothelial growth factor (VEGF) is a significant regulator of cancer angiogenesis, it is essential to develop a technology for its sensitive detection. Herein, we sensitized a chemiluminescence (CL) immunoassay through the combination of H2O2-sensitive TGA-CdTe quantum dot (QD) as signal transduction, dextran as a cross-linker to prepare enzyme-labeled antigen and the ultrahigh bioactivity of catalase (CAT) as reporter enzyme. RESULTS: Under the optimized experimental conditions, the chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA) method can detect VEGF in the excellent linear range of 2-35,000 pg mL-1, with a detection limit (S/N = 3) of 0.5 pg mL-1 which was approximately ten times lower than the commercial colorimetric immunoassay. This proposed method has been successfully applied to the clinical determination of VEGF in the human serum samples, and the results illustrated an excellent correlation with the conventional ELISA method (R2 = 0.997). The suitable recovery rate of the method in the serum ranged from 97 to 107%, with a relative standard deviation of 1.2% to 13.4%. CONCLUSIONS: The novel immunoassay proposes a highly sensitive, specific, and stable method for very low levels detection of VEGF that can be used in the primary diagnosis of tumors. With the well-designed sensing platform, this approach has a broad potential to be applied for quantitative analysis of numerous disease-related protein biomarkers for which antibodies are available.

Down Regulation of ackA-pta Pathway in Escherichia coli BL21 (DE3): A Step Toward Optimized Recombinant Protein Expression System.

BACKGROUND: One of the most important problems in production of recombinant protein is to attain over-expression of the target gene and high cell density. In such conditions, the secondary metabolites of bacteria become toxic for the medium and cause cells to die. One of these aforementioned metabolites is acetate, which enormously accumulated in the medium, so that both cell and protein yields are affected. OBJECTIVES: To overcome this problem, several strategies applied. In this research we used antisense RNA strategy, where the transcription of phosphotransacetylase (PTA) and acetate kinase (ACK), two acetate pathway key enzymes, could be controlled, which led to reduced acetate production. MATERIALS AND METHODS: In order to achieve this, recombinant plasmid harboring antisense sequences targeting both of pta and ackA was assembled, after transfecting to the cells, its effects on the cell growth and acetate accumulation in the minimal media was assessed and compared with the control, the plasmid without antisense cassette, in presence and absence of IPTG in Escherichia coli BL21 (DE3). RESULTS: It was observed that the mentioned strategy partially affect the growth and amount of excreted acetate in comparison with the control. In addition it was found that high down-regulation of the acetate production pathway reduces the growth rate of E. coli BL21 (DE3). CONCLUSIONS: The study principally proved the importance of this strategy in acetate excretion control.

Synthesis of a unique high-performance poly-horseradish peroxidase complex to enhance sensitivity of immunodetection systems.

Because early detection is the first step in successful therapy, increasing the sensitivity of detection systems has always been considered as one of the major trends in development of these technologies. Therefore, we have fabricated a high-performance poly-horseradish peroxidase (HRP) complex and analyzed it in different formats of immunodetection systems. To construct this complex, dextran-aldehyde was prepared by oxidation of dextran in the presence of sodium periodate. Activated polymer was then coupled to lysine amino acids and accomplishment of the process was evaluated with trinitrobenzenesulfonic acid. Following conjugation of HRP to free amino groups of lysine, the stage's accuracy and the rate of conjugation were demonstrated by SDS-PAGE. Then, conjugation of poly-HRP complex to streptavidin by biotin was performed. The results of a series of experiments confirmed the complete synthesis of streptavidin-poly-HRP complex by this procedure. Finally, we compared our harvested complex with the golden standard complex available for ELISA and immunohistochemistry (IHC). The results showed the high efficiency of the synthesized complex. Consequently, this complex can be applicable in highly sensitive detection technologies. Conjugating this complex to any antibody by using biotin-streptavidin bridging and preparing poly-HRP-labeled antibodies will be a valuable multifold approach to increase the sensitivity of detection systems, which can be applicable in ELISA, immunocytochemistry, and IHC methods.

Investigation in vitro Expression of CatSper Sub Fragment followed by Production of Polyclonal Antibody: Potential Candidate for The Next Generation of Non Hormonal Contraceptive.

OBJECTIVE: CatSper is a voltage-sensitive calcium channel that is specifically expressed in the testis and it has a significant role in sperm performance. CatSper (1-4) ion channel subunit genes, causes sperm cell hyperactivation and male fertility. In this study, we have explored targeting of the extracellular loop as an approach for the generation of antibodies with the potential ability to block the ion channel and applicable method to the next generation of non-hormonal contraceptive. MATERIALS AND METHODS: In this experimental study, a small extracellular fragment of CatSper1 channel was cloned in pET-32a and pEGFP-N1 plasmids. Then, subsequent methods were performed to evaluate production of antibody: 1) pEGFP-N1/CatSper was used as a DNA vaccine to immunize Balb/c mice, 2) The purified protein of pET-32a/CatSper was used as an antigen in an enzyme-linked immunosorbent assay (ELISA) and western- blot, and 3) The serum of Balb/-c mice was used as an antibody in ELISA and western-blot. The statistical analysis was performed using the Mann Whitney test. RESULTS: The results showed that vaccination of the experimental group with DNA vaccine caused to produce antibody with (p<0.05) unlike the control group. This antibody extracted from Balb/c serum could recognize the antigen, and it may be used potentially as a male contraception to prevent sperm motility. CONCLUSION: CatSpers are the promising targets to develop male contraceptive because they are designed highly specific for sperm; although, no antagonists of these channels have been reported in the literature to date. As results showed, this antibody can be used in male for blocking CatSper channel and it has the potential ability to use as a contraceptive.

Generation of a novel high-affinity monoclonal antibody with conformational recognition epitope on human IgM.

As IgM is the first isotype of antibody which appears in blood after initial exposure to a foreign antigen in the pattern of primary response, detection, and quantification of this molecule in blood seems invaluable. To approach these goals, generation, and characterization of a highly specific mAb (monoclonal antibody) against human IgM were investigated. Human IgM immunoglobulins were used to immunize Balb/c mice. Spleen cells taken from the immunized animals were fused with SP2/O myeloma cells using PEG (polyethylene glycol, MW 1450) as fusogen. The hybridomas were cultured in HAT containing medium and supernatants from the growing hybrids were screened by enzyme-linked immunosorbent assay (ELISA) using plates coated with pure human IgM and the positive wells were then cloned at limiting dilutions. The best clone designated as MAN-1, was injected intraperitoneally to some Pristane-injected mice. Anti-IgM mAb was purified from the animals' ascitic fluid by protein-G sepharose followed by DEAE-cellulose ion exchange chromatography. MAN-1 interacted with human IgM with a very high specificity and affinity. The purity of the sample was tested by SDS-PAGE and the affinity constant was measured (K(a) = 3.5 x 10(9)M(-1). Immunoblotting and competitive ELISA were done and the results showed that the harvested antibody recognizes a conformational epitope on the mu chain of human IgM and there was no cross-reactivity with other subclasses of immunoglobulins. Furthermore, isotyping test was done and the results showed the subclass of the obtained mAb which was IgG(1)kappa.

The effect of wild type P53 gene transfer on growth properties and tumorigenicity of PANC-1 tumor cell line.

BACKGROUND: The p53 protein function is essential for the maintenance of the nontumorigenic cell phenotype. Pancreatic tumor cells show a very high frequency of p53 mutation. To determine if restoration of wild type p53 function can be used to eliminate the tumorigenic phenotype in these cells, pancreatic tumor cell lines, PANC-1 and HTB80, differing in p53 status were stably transfected with exogenous wild type p53 gene. METHODS: The transfection was performed using Polybrene/DMSO-Assisted Gene Transfer method. The wild type p53 gene integration into genomic DNA was detected by Southern blot and PCR. Furthermore, the expression of wild type p53 protein was detected in selected clones by immunohistochemistry and Western blot. RESULTS: While HTB80 cell line failed to produce a stable p53 expressing clone, the PANC-1 cells produced stable lines. Following characterization of clones, the growth rate and tumorigenicity of PANC-1 wild type p53 clones were compared to the control cells. Our data showed that the expression of wild type p53 decreased the growth rate of PANC-1 cells. It was also observed that the expression of wild type p53 in PANC-1 cells suppressed its potential for tumor formation in nude mice, completely, while the parental line leads to the formation of a relatively large tumor. CONCLUSION: Our results suggest that gene therapy based on restoration of wild type p53 protein function in pancreatic tumor cells with high amount of mutant p53 is a feasible option in pancreatic cancer treatment.

A novel monoclonal antibody with catalytic activity against beta human chorionic gonadotropin.

In this study, we report for the first time, production of monoclonal antibody (MAb) against beta human chorionic gonadotropin (hCG) with proteolytic activity. MAb "7D9" was raised in Balb/C mice using purified human chorionic gonadotropin. Immunoblot analysis and enzyme-linked immunoabsorbent assay (ELISA) showed that this MAb reacts with beta hCG. The epitope for this antibody appears to be located in the C-terminal of beta chain as suggested by the absence of cross-reaction with other glycoprotein hormones such as FSH, TSH and LH. Our data reveal that this MAb is very unstable and has autodegradation characteristics. Zymogram analyses also show that 7D9 MAb has a high level of hydrolytic activity against different substrates such as casein and gelatin. This proteolytic activity can be inhibited by EDTA. These findings demonstrate the proteolytic character of 7D9 MAb and consequently explain its instability.

Efficacy of dendrimer-mediated angiostatin and TIMP-2 gene delivery on inhibition of tumor growth and angiogenesis: in vitro and in vivo studies.

Gene transfer is an attractive approach to fight cancer by targeting cancer cells or their vasculature. Our study reports the inhibition of tumor growth and angiogenesis by a nonviral method using dendrimers associated with 36-mer anionic oligomers (ON36) for delivering angiostatin (Kringle 1-3) and tissue inhibitor of metalloproteinase (TIMP)-2 genes. The optimal concentrations of dendrimers and ON36 for an efficient green fluorescent protein (GFP) plasmid delivery in endothelial cells (HMEC-1) and cancer cells (MDA-MB-435) were first chosen. Then the efficacy of transfection was determined by testing angiostatin and TIMP-2 secretion by Western blot and the biologic effects were evaluated. Angiostatin gene transfer markedly reduced in vitro (i) HMEC-1 but not MDA-MB-435 proliferation; (ii) HMEC-1 and MDA-MB-435 wound healing reparation; and (iii) capillary tube formation. TIMP-2 gene transfer did not affect cell proliferation but strongly inhibited (i) wound healing of HMEC-1 and MDA-MB-435 cells; and (ii) capillary tube formation. Supernatants of transfected-MDA-MB-435 cells also inhibited the formation of angiogenic networks on Matrigel, indicating a paracrine effect. In vivo, intratumoral angiostatin or TIMP-2 gene delivery using dendrimers associated with ON36 effectively inhibited tumor growth by 71% and 84%, respectively. Combined gene transfer resulted in 96% inhibition of tumor growth. Tumor-associated vascularization was also greatly reduced. These findings provide a basis for the further development of nonviral delivery of genes to fight cancer.