Fabrication of Antibody Conjugated Super Magnetic Oxide Nanoparticles for Early Detection of Prostate Cancer.

BACKGROUND: Prostate cancer is one of the most widespread cancers in the world. Early diagnosis is the most important factor in treatment efficiency. Furthermore, new methods for early diagnosis and treatment play an important role. In this study, we designed targeted conjugation of antibodies with iron nanoparticles and evaluated the binding properties of antibodies to prostate cancers and benign tissues. This method in addition to having a lower cost has high sensitivity and specificity. METHODS: Anti- PSCA antibodies were purified and conjugated to super magnetic oxide nanoparticles (SPION). Then, iron staining on prostate adenocarcinoma tissues was performed. At the same time, immunohistochemically staining was performed on similar tissues to compare the results. In addition, benign prostatic hyperplasia (BPH) samples were used as a control sample. RESULTS: In adenocarcinoma tissues with iron staining, many blue spots are seen compared to benign tissues, and the number of these spots increases with increasing tumor grade. CONCLUSION: These findings indicate the characteristic of iron staining as a conjugate antibody to iron can be an appropriate approach to specific staining of tumor markers in cancer tissues and can be used to diagnose prostate cancer due to its safety, low cost, sensitivity, and specificity.

Production and Conjugation of Truncated Recombinant Diphtheria Toxin to VEGFR-2 Specific Nanobody and Evaluation of its Cytotoxic Effect on PC-3 Cell Line.

Immunotoxins have represented a great potency in targeted therapeutics to encounter tumors. They consist of a protein toxin conjugated to a targeting moiety, which recognizes a specific antigen on surface of cancer cells and accordingly induces cell death by toxin segment. The targeting part could be a nanobody, which is a group of antibodies composed of an only functional single variable heavy chain (VHH).Therefore, this study was done to produce an immunotoxin (VGRNb-DT) by chemical conjugation of a truncated diphtheria toxin moiety to an anti-vascular endothelial growth factor receptor 2(VEGFR-2) nanobody, and to identify effectiveness of immunotoxin in recognizing the VEGFR-2- positive cancer cells and inhibiting cell growth and survival. Diphtheria toxin was expressed and purified by nickel affinity chromatography, and accordingly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis confirmed its expression. Function of heterobifunctional crosslinkers, Sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate), and SATP (N-succinimidyl-S- acetylthiopropionate) for bioconjugation purposes was acknowledged by cation exchange high-performance liquid chromatography (HPLC). Cytotoxicity of immunotoxin was evaluated on the VEGFR-2 positive PC-3 cell line by MTT assay. Overexpression of VEGFR-2 in the PC-3 cell line allowed immunotoxin to recognize them by anti-VEGFR-2 nanobodies. The concentrations above 5 µg/ml represented a significant decrease in cell survival rate in PC-3 cells compared to HEK293 cells (VEGFR-2 negative cells) as controls.VGRNb-DT demonstrated a successful bioconjugation; furthermore, variable concentrations were correlated with cell death in prostate cancer PC-3 cells.

Effects of synthetic silymarin-PLGA nanoparticles on M2 polarization and inflammatory cytokines in LPS-treated murine peritoneal macrophages.

OBJECTIVES: Silymarin (SM) is a natural antioxidant compound with good anti-inflammatory effects, but its poor water solubility restricts its usage. Today, nanomaterial compounds (such as PLGA Poly D, L-lactic-co-glycolic acid) can provide a proper drug delivery system and help improve the accessibility of bioactive compounds to cells and tissues. MATERIALS AND METHODS: In this study, PLGA nanoparticles (NPs) containing SM (SM-PLGA) were synthesized and characterized and their biological effects were evaluated on M2 macrophage polarization to regulate inflammation. SM-PLGA NPs were fabricated by the oil in water emulsion (O/W) method. Macrophages (MQs) were isolated from mouse peritoneum by the cold RPMI lavage protocol. Primary mouse MQ cells were treated by SM and SM-PLGA NPs and then stimulated with lipopolysaccharide (LPS). M2 polarization was evaluated by measurements of cytokine secretion levels (TNF-α, IL1-ß, and IL-10), flow cytometry markers (F4/80, CD11b, CD38, and CD206), and the expression of specific proteins (M2 Ym1 and Fizz1). RESULTS: SM-PLGA characterization showed that NPs were fabricated in the desired form. SM and SM-PLGA decreased pro-inflammatory cytokines (TNF-α and IL1-ß) and increased IL10 as an anti-inflammatory cytokine. On the other hand, the M2-associated markers and proteins increased following treatment with SM and SM-PLGA. Post-hoc analysis indicated that these changes were more pronounced in the SM-PLGA group. CONCLUSION: This study revealed that SM-PLGA could markedly promote M2 polarization, thereby providing a valuable medical approach against sepsis and septic shock.

Extraction, Chemical Composition, Antioxidant Property, and In-vitro Anticancer Activity of Silymarin from Silybum marianum on Kb and A549 Cell Lines.

INTRODUCTION: This study aimed to evaluate the antioxidant property of Silymarin (SM) extracted from the seed of Silybum marianum and its anticancer activity on KB and A549 cell lines following 24, 48, and 72 h of treatment. METHODS: Ten grams of powdered S. marianum seeds were defatted using n-hexane for 6 hours and then extracted by methanol. The Silymarin extracted of extraction components. The extracted components of Silymarin were measured by spectrophotometric assay and HPLC analysis. 2, 2- diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, phenol content, total flavonoid content, and total antioxidant capacity were measured to detect the antioxidant properties of SM. The anticancer activity of the SM on cell lines evaluated by MTT. RESULTS: In HPLC analysis, more than 50% of the peaks were related to silybin A and B. SM was reduced DPPH (the stable free radical) with a 50% inhibitory concentration (IC50) of 6.56 µg/ ml in comparison with butylated hydroxyl toluene (BHT), which indicated an IC50 of ~3.9 µg/ ml. The cytotoxicity effect of SM on the cell lines was studied by MTT assay. The cytotoxicity effect of the extracted Silymarin on KB and A549 cell lines was observed up to 80 and 70% at 156 and 78 µg/ml, respectively. The IC50 value of the extracted SM on KB and A549 cell lines after 24 hours of treatment was seen at 555 and 511 µg/ml, respectively. CONCLUSION: Due to the good antioxidant and anticancer properties of the isolated Silymarin, its use as an anticancer drug is suggested.

Designing a chimeric subunit vaccine for influenza virus, based on HA2, M2e and CTxB: a bioinformatics study.

BACKGROUND: Type A influenza viruses are contagious and even life-threatening if left untreated. So far, no broadly protective vaccine is available due to rapid antigenic changes and emergence of new subtypes of influenza virus. In this study, we exploited bioinformatics tools in order to design a subunit chimeric vaccine from the antigenic and highly conserved regions of HA and M2 proteins of H7N9 subtype of influenza virus. We used mucosal adjuvant candidates, including CTxB, STxB, ASP-1, and LTB to stimulate mucosal immunity and analyzed the combination of HA2, M2e, and the adjuvant. Furthermore, to improve the antigen function and to maintain their three-dimensional structure, 12 different linkers including six rigid linkers and six flexible linkers were used. The 3D structure model was generated using a combination of homology and ab initio modeling methods and the molecular dynamics of the model were analyzed, either. RESULTS: Analysis of different adjuvants showed that using CtxB as an adjuvant, results in higher overall vaccine stability and higher half-life among four adjuvant candidates. Fusion of antigens and the CTxB in the form of M2e-linker-CTxB-linker-HA2 has the most stability and half life compared to other combination forms. Furthermore, the KPKPKP rigid linker showed the best result for this candidate vaccine among 12 analyzed linkers. The changes in the vaccine 3D structure made by linker insertion found to be negligible, however, although small, the linker insertion between the antigens causes the structure to change slightly. Eventually, using predictive tools such as Ellipro, NetMHCpan I and II, CD4episcore, CTLpred, BepiPred and other epitope analyzing tools, we analyzed the conformational and linear epitopes of the vaccine. The solubility, proteasome cleavage sites, peptidase and potential chemical cutters, codon optimization, post translational modification were also carried out on the final vaccine. CONCLUSIONS: It is concluded that M2e-Linker-CTxB-Linker-HA2 combination of chimeric vaccine retains its 3D structure and antigenicity when KPKPKP used as linker and CTxB used as adjuvant.

Preparation of Diphtheria and Pseudomonas Exotoxin A Immunotoxins and Evaluation of Their Cytotoxicity Effect on SK-BR-3, BT-474, and MDA-MB-231 Breast Cancer Cell Lines.

Immunotoxin targeted therapy is a promising way of cancer therapy that is made from a toxin attached to an antibody which target a specific protein presented on cancer cells. In this study, we introduce immunotoxins comprising of truncated pseudomonas exotoxin A (PEA) and diphtheria toxin (DT) conjugated to trastuzumab. The effectiveness of 20 and 30 µg/ml immunotoxins and trastuzumab were studied on SK-BR-3 and BT-474 HER2/neu positive breast cancer cell lines by a cell death assay test. The produced immunotoxins have the potential to reduce the therapeutic dose of the trastuzumab and in the same time achieve higher efficiency.

Cloning and expression of NS3 helicase fragment of hepatitis C virus and the study of its immunoreactivity in HCV infected patients.

OBJECTIVES: Hepatitis C is a major cause of liver failure worldwide. Current therapies applied for this disease are not fully effective and produce side effects in most cases. Non-structural protein 3 helicase (NS3) of HCV is one of the key enzymes in viral replication and infection. Therefore, this region is a promising target to design new drugs and therapies against HCV infection. The aim of this study was cloning and expression of HCV NS3 helicase fragment in Escherichia coli BL21 (DE3) using pET102/D-TOPO expression vector and studying immunoreactivity of the expressed antigen in Iranian infected with hepatitis C. MATERIALS AND METHODS: The viral RNA was extracted from the serum of HCV infected patient. The NS3 helicase region was amplified by RT-PCR. The PCR product was directionally cloned into the expression vector pET102/D-TOPO and transformed into the BL21 strain of E. coli (DE3). The transformed bacteria were then induced by adding 1mM isopropyl-ß-D-thiogalactopyranoside (IPTG) into the culture medium to enhance the protein expression. SDS-PAGE and western blotting were carried out to identify the protein under investigation, and finally purified recombinant fusion protein was used as the antigen for ELISA method. RESULTS: The insertion of the DNA fragment of the NS3 region into the expression vector was further confirmed by PCR and sequencing. SDS-PAGE analysis showed the successful expression of the recombinant protein of interest. Furthermore, immunoreactivity of fusion NS3 helicase was confirmed by ELISA and western blotting. CONCLUSION: It seems that this recombinant protein could be a useful source of antigen for future studies on HCV diagnosis and therapy.

Immunoreactivity assessment of hepatitis C virus NS3 protease and NS5A proteins expressed in TOPO cloning system.

BACKGROUND: Hepatitis C virus (HCV) is a major cause of acute and chronic liver disease. Numerous screening assays based on the detection of immunoresponses to HCV structural and nonstructural proteins have been designed. Various studies have demonstrated genotype-specific differences in anti-HCV antibody responses to different HCV proteins. METHODS: Full-length NS3 protease and N-terminally truncated NS5A were expressed using pET TOPO 102/D system. Antigenicity of the purified recombinant proteins was assessed by immunoblotting and indirect enzyme-linked immunosorbent assay (ELISA). Furthermore, anti-HCV antibody responses to the recombinant proteins were evaluated in three prevalent genotypes in Iran. RESULTS: We were able to express and purify NS5A and NS3 protease using TOPO cloning system. The HCV NS3 protease and NS5A produced in BL21 Star (DE3) was immunoreactive. Our results demonstrate that NS3 protease and NS5A have good immunoreactivity, but they are not sufficient for detecting all HCV-positive sera. No significant genotype-specific differences were detected in immunoresponses to the recombinant proteins. CONCLUSION: In conclusion, we successfully isolated, expressed, and purified substantial amount of HCV NS3 protease and N-terminally truncated NS5A, and used them as capturing antigens in a screening ELISA assay with high sensitivity, reproducibility, and specificity. Accordingly, it is well confirmed that TOPO cloning system can be used as a dynamic system in order to express higher amount of immunoreactive viral proteins.

Recombinant proteins: hopes for tissue engineering.

Proteins constitute a group of key molecules with many applications in tissue engineering. Use of proteins provided from natural sources has several limitations which are overcome by the use of recombinant proteins. So far, the recombinant forms of many proteins with tissue engineering applications have been developed including structural proteins, growth factors and cytokines. This technology has enabled the development of specifically designed proteins such as growth factors with matrix binding domains, and hybrid structural proteins with improved mechanical properties. Recombinant proteins are produced either ex vivo or in vivo, by local gene therapy protocols, and are of medical and economic benefits. Due to the high applicability of recombinant proteins in tissue engineering, it is recommended to include this platform as an infrastructural element in any tissue engineering program.