Docetaxel-loaded nanostructured lipid carriers functionalized with trastuzumab (Herceptin) for HER2-positive breast cancer cells.

The aim of the present study was to prepare Herceptin targeted nanostructured lipid carriers (NLCs) of docetaxel (DTX). Herceptin was conjugated by chemical and physical methods to NLCs prepared by solvent extraction technique followed by probe sonication. Different types of fatty amines were used in construction of NLCs. The NLCs were characterized for their antibody coupling efficiency, particle size, zeta potential, polydispersity index, drug entrapment efficiency and drug release profiles. The toxicity of NLCs on MDA-MB-468 (HER2 negative receptor) and BT-474 (HER2 positive) breast cancer cell lines was evaluated by MTT assay. Also their cellular uptake was studied by flow-cytometry and fluorescent microscopy. The results showed the NLCs containing stearyl amine had the lowest particle size, the highest zeta potential and antibody coupling efficiency values. Herceptin binding to NLCs led to reduction in zeta potential and drug entrapment efficiency while, particle size increased. The NLCs containing spermine(SP) released DTX slower than other fatty amines. Non-conjugated nanoparticles containing DTX had more toxicity than the free DTX on both cell lines. Herceptin targeted NLCs caused more mortality on BT-474 cells than MDA-MB-468 cells. Flow-cytometry studies revealed enhanced cellular uptake of nanoparticles chemically conjugated by Herceptin on the BT-474 cells. DTX loaded in chemically conjugated NLCs to Herceptin showed more cytotoxic effects than the physically coated nanoparticles. The Herceptin conjugated NLCs seem promising in oriented delivery of DTX to HER2 positive breast cancer cells.

Cytotoxic and Apoptotic Effects of Three Types of Silver-Iron Oxide Binary Hybrid Nanoparticles.

BACKGROUND: Multifunctional nanostructures have received great deal of attention in biomedical area due to their capabilities in the development of new therapeutic and diagnostic agents. Silver and iron oxide nanoparticles, owing to their specific characteristics, are considered to develop bifunctional hybrid nanoparticles for magnetic delivery of silver nanoparticles as cytotoxic agent toward cancer cells. OBJECTIVE: This study was designed to explore the in-vitro cytotoxic and apoptotic activities of three different silver-iron oxide binary hybrid nanoparticles on human liver hepatocellular carcinoma cell line. METHOD: Three different silver-iron oxide binary hybrid nanoparticles were synthesized and characterized through the designed procedures. Apoptosis induction was investigated through flow cytometry and the influence on bax gene expression level was analyzed using quantitative real time polymerase chain reaction. RESULTS: All the three types of silver-iron oxide hybrid nanoparticles (possessing different characteristics) exhibited cytotoxic and apoptotic effects. Furthermore, the up regulation of bax gene expression suggested the involvement of the intrinsic pathway of apoptosis. Some of the transcription regulators which could interact with bax gene promoter were analyzed and found out to be mostly contributed in the stress responses. Among the test nanoparticles, the strongest cytotoxic and apoptotic effect was induced by the binary hybrid nanoparticle which was synthesized with glucose as reducing agent; suggesting that the biological activity was affected by different characteristics of the designed nanoparticles. CONCLUSION: Combined properties of silver and magnetic nanoparticles in the binary hybrid nanoparticles, provide a great potential to be exploited in the cancer therapy, where the combination of cytotoxicity and magnetic targeting is desired.

Synthesis of molecular imprinted polymers for selective extraction of domperidone from human serum using high performance liquid chromatography with fluorescence detection.

In this study a novel method is described for selective quantization of domperidone in biological matrices applying molecular imprinted polymers (MIPs) as a sample clean up procedure using high performance liquid chromatography coupled with a fluorescence detector. MIPs were synthesized with chloroform as the porogen, ethylene glycol dimethacrylate as the crosslinker, methacrylic acid as the monomer, and domperidone as the template molecule. The new imprinted polymer was used as a molecular sorbent for separation of domperidone from serum. Molecular recognition properties, binding capacity and selectivity of MIPs were determined. The results demonstrated exceptional affinity for domperidone in biological fluids. The domperidone analytical method using MIPs was verified according to validation parameters, such as selectivity, linearity (5-80ng/mL, r(2)=0.9977), precision and accuracy (10-40ng/mL, intra-day=1.7-5.1%, inter-day=4.5-5.9%, and accuracy 89.07-98.9%).The limit of detection (LOD) and quantization (LOQ) of domperidone was 0.0279 and 0.092ng/mL, respectively. The simplicity and suitable validation parameters makes this a highly valuable selective bioequivalence method for domperidone analysis in human serum.

Facile fabrication of uniform hollow silica microspheres using a novel biological template.

Biological templates, due to their ease of preparation and surface modifications can be a promising approach to fabricate hollow structures. In this study, for the first time, a biological template was used for the production of hollow silica microspheres. Silica was successfully deposited on Staphylococcus aureus cells surface using the Stöber method. The hollow silica spheres with a mesoporous shell of approximately 700nm in diameter were produced by applying this novel method.

Experimental design of medium optimization for invertase production by Pichia sp.

The culture medium requirement for invertase production by Pichia sp. was optimized and identified by initial screening method of Plackett-Burman. Furthermore, optimum concentrations of medium components, which were selected by in initial screening by Plackett-Burman, were determined by the Box-Behnken and its representative three-factor response-surface method. The regression models showed significantly high R (2) values of 97% for invertase activities, indicating that they are appropriate for predicting relationships between yeast extract, peptone and sucrose concentration with invertase production. According to the model the optimal concentrations of sucrose, yeast extract and peptone were 40, 5 and 4 g/ml, respectively. These predicted conditions were verified by validation experiments. In the optimized medium Pichia sp. produced invertase with activity of 38.71 U/ml, which is 4 times higher than that produced in original medium. Thus, this statistical approach enabled rapid identification and integration of key medium parameters for Pichia sp. BCCS M1, resulted the high invertase production.

Isolation, identification, and media optimization of high-level cellulase production by Bacillus sp. BCCS A3, in a fermentation system using response surface methodology.

Cellulases are important glycosyl hydrolase enzymes, which break down cellulose to ß-glucose. They have been used widely in biotechnological processing such as bioethanol production. In this work we studied maximizing cellulase production by Bacillus sp. BCCS A3 using response surface methodology (RSM). A good result was attained with these conditions (% w/v): tryptone 0.1, Na2PO4 0.25, (NH4)2SO4 0.2, MgSO4 · 7H2O 0.005, CaCl2 0.005, KH2PO4 0.1, NaCl 0.1, sodium carboxymethylcellulose (CMC) 0.75, and pH 9. The cellulase activity in optimized medium was 49.80 U/ml. Moreover, high level of enzyme production was obtained by using fermentor system (50.30 U/ml). Thus, according to the obtained results, this statistical method provided quick identification and integration of key medium details for Bacillus sp. BCCS A3, leading to more cellulase production.

Prevention and Inhibition of TC-1 Cell Growth in Tumor Bearing Mice by HPV16 E7 Protein in Fusion with Shiga Toxin B-Subunit from shigella dysenteriae.

OBJECTIVE: For immunotherapy of human papillomavirus (HPV) -16-associated cervical cancers the E7 protein is considered a prime candidate. However it is a poor inducer of cytotoxic T-cell response, when being used as a singular antigen in protein vaccination. Hence, in this study we focused on the utilization of a vaccine delivery system for prevention or treatment of cervical cancer. MATERIALS AND METHODS: In this experimental study, we designed and evaluated a novel fusion protein comprising HPV16 E7 antigen fused to Shiga toxin B-subunit (STxB) as both an antigen vector and an adjuvant. Then we designed two preventive and therapeutic tumor models to investigate the prevention and inhibition of TC-1 cell growth in female C57BL/6 mice, respectively. In each model, mice were immunized with the recombinant protein of E7-STxB or E7 without any adjuvant. RESULTS: We demonstrated that prophylactic immunization of E7-STxB protected mice against TC-1 cells. Also in the therapeutic model, E7-STxB inhibited TC-1 tumor growth inlungs. The results were significant when compared with the immunization of E7 singularly. CONCLUSION: We concluded that immunization with the E7-STxB protein without any adjuvant could generate anti-tumor effect in mice challenged with TC-1 cells.This research verifies the clinical applications and the future prospects of developing HPV16 E7 therapeutic vaccines fused to immunoadjuvants.

Induction of antitumor immunity against cervical cancer by protein HPV-16 E7 in fusion with ricin B chain in tumor-bearing mice.

OBJECTIVE: In immunotherapy of HPV-16-associated cervical cancers, the E7 protein is considered as a prime candidate. However, it is a poor inducer of a cytotoxic T-cell response when used as a singular antigen in protein vaccination. Therefore, to design effective cancer vaccines, the best tumor antigens should be combined with the most effective immunogens or drug delivery tools to achieve positive clinical results. In this study, we fused HPV-16 E7 with the lectin subunit of ricin toxin (RTB) from castor plant as a vaccine adjuvant/carrier. MATERIALS AND METHODS: After reaching the soluble form of the recombinant protein, we designed 2 preventive and inhibition tumor models for investigation of the prevention and rejection of TC-1 cell growth in female C57BL/6 mice, respectively. In each model, mice were immunized with the recombinant protein of E7-RTB or E7 without any adjuvant. RESULTS: We demonstrated that prophylactic immunization of E7-RTB protected mice against challenge from TC-1 cells. Also in the therapeutic model, E7-RTB could inhibit TC-1 tumor growth in the lung. The results were significant compared with the immunization of E7 singularly. CONCLUSIONS: We concluded that immunization with E7-RTB protein without any adjuvant could generate antitumor effects in mice challenged with TC-1 cells. This research verifies the clinical applications and the future prospects for development of HPV-16 E7 therapeutic vaccines fused to immunoadjuvants.

Modification in media composition to obtain secretory production of STxB-based vaccines using Escherichia coli.

Shiga toxin B-subunit (STxB) from Shigella dysenteriae targets in vivo antigen to cancer cells, dendritic cells (DC) and B cells, which preferentially express the globotriaosylceramide (Gb3) receptor. This pivotal role has encouraged scientists to investigate fusing STxB with other clinical antigens. Due to the challenges of obtaining a functional soluble form of the recombinant STxB, such as formation of inclusion bodies during protein expression, scientists tend to combine STxB with vaccine candidates rather than using their genetically fused forms. In this work, we fused HPV16 E7 as a vaccine candidate to the recombinantly-produced STxB. To minimize the formation of inclusion bodies, we investigated a number of conditions during the expression procedure. Then various strategies were used in order to obtain high yield of soluble recombinant protein from E. coli which included the use of different host strains, reduction of cultivation temperature, as well as using different concentrations of IPTG and different additives (Glycin, Triton X-100, ZnCl(2)). Our study demonstrated the importance of optimizing incubation parameters for recombinant protein expression in E. coli; also showed that the secretion production can be achieved over the course of a few hours when using additives such as glycine and Triton X-100. Interestingly, it was shown that when the culture mediums were supplemented by additives, there was an inverse ratio between time of induction (TOI) and the level of secreted protein at lower temperatures. This study determines the optimal conditions for high yield soluble E7-STxB expression and subsequently facilitates reaching a functionally soluble form of STxB-based vaccines, which can be considered as a potent vaccine candidate for cervical cancer.

Preparation of novel magnetic fluorescent nanoparticles using amino acids.

Nanotechnology has opened new gates to pharmaceutical sciences and medicine from the aspect of drug delivery and imaging systems. Currently, bimodal fluorescent-magnetic nanoparticles are of great interest to biomedical scientists. In order to constructing these kinds of nanoparticles, fluorescent molecules should be linked to a magnetic core, while luminescence quenching is prevented. In order to alleviate this effect, usually fluorescent molecules are attached to a magnetic core after a multistep hydrocarbon, polymer or silica coating, which significantly increases the particle's size and reduces its magnetic saturation value. In this study, for the first time, amino acids (L-lysine and L-arginine) have been used as a linker and spacer between a fluorescent molecule (FITC) and a magnetic nanoparticle (Fe(3)O(4)) in a simple, two-step synthesis. Also, 3-aminopropyltriethoxysilane (APTES) was used without any previous silica coating for fluorescent magnetic nanoparticles construction. Routinely APTES is used after silica coating by tetraethoxysilane (TEOS). Either of L-lysine, L-arginine and APTES coating provides surface functional groups which interact with the isothiocyanate group of FITC. According to the obtained results, amino acids could be used for successful construction of fluorescent magnetic nanoparticles in a simple synthesis pathway, without any significant impact on the excitation and emission properties of fluorescent molecule.

The possible role of HSPs on Behçet's disease: a bioinformatic approach.

Current evidence lends increasing support to immunoinflammatory mechanisms as one of the prime pathogenic processes involved in the development and progression of Behçet's disease (BD). It has been observed that most human beings have cellular and humoral reactions against microbial heat shock proteins (HSPs). The observation that eukaryotic and prokaryotic HSPs have high sequence similarity promoted the hypothesis that HSPs might be potential candidates for molecular mimicry and could act as potentially dangerous autoantigens. In this study, using bioinformatics tools, we examined the hypothesis that HSPs (evolutionarily conserved proteins), which are present in pathogenic and commensal organisms and their hosts, provide the stimulus that initiates BD in susceptible individuals. In this regards, the nucleotide and amino acid sequences of the human HSP 60 kDa and bacterial HSP 60 kDa deposited in the NCBI and PDB databases were subjected to analysis using bioinformatics tools, including The CLC Sequence Viewer and MEGA softwares. These data showed that the sequence homology between bacterial and self HSPs (leading to cross-reactivity and molecular mimicry phenomenon) may be associated with the development of the disease; and suggesting that microbial HSPs, which cross-react with host tissues and elicit significant immune responses are possible pathogenetic agents involved in the development and progression of BD.

THE BIOTRANSFORMATION, BIODEGRADATION, AND BIOREMEDIATION OF ORGANIC COMPOUNDS BY MICROALGAE(1).

Rapid growth in the biotechnological industry and production has put tremendous pressure on the biological methods that may be used according to the guidelines of green chemistry. However, despite continuing dramatic increases in published research on organic biotransformation by microorganisms, more research exists with microalgae. Our efforts in transforming chemicals such as organic compounds for the production of functionalized products help to lessen the environmental effects of organic synthesis. These biotransformations convert organic contaminants to obtain carbon or energy for growth or as cosubstrates. This review aims to focus on the potential of microalgae in transformation, conversion, remediation, accumulation, degradation, and synthesis of various organic compounds. However, these technologies have the ability to provide the most efficient and environmentally safe approach for inexpensive biotransforming of a variety of organic contaminants, which are most industrial residues. In addition, the recent advances in microalgal bioactivity were discussed.

l-Asparaginase Production by Moderate Halophilic Bacteria Isolated from Maharloo Salt Lake.

l-Asparaginase is an anti-neoplastic drug used in lymphoblastic leukemia chemotherapy. Nowadays, this enzyme derived from bacterial sources, mostly l-asparaginase II from Escherichia coli and in lesser amount l-asparaginase of Erwinia sp. has medical utilization. The long-term usage of these agents leads to allergic reactions and new asparaginase with new immunological characteristics is required. Halophilic bacteria might contain l-asparaginase with novel immunological properties that can be used in hypersensitive patients. In this experiment, we have screened moderate Halophilic bacteria for l-asparaginase production ability and showed that Halophilic bacteria produce intra- and extracellular l-asparaginase. Bacillus sp. BCCS 034 was found to produce the highest l-asparaginase (1.64 IU/ml supernatant) extracellularly.

Chlamydomonas as a "new" organism for biodiesel production.

The production of biodiesel from a naturally isolated strain of Chlamydomonas was investigated. The microalgal strain was isolated from the rice paddy-field soil samples during a screening program. The identification was done using physiological and molecular approaches. After reaching the stationary phase of growth, the total content of the lipids was extracted. The extracted fatty acids were primarily esterified and then identified through TLC and GC/MS analysis. Several types of fatty acid methyl esters (FAMEs) were identified in the isolated microalga and the presence of at least nine FAMEs in Chlamydomonas sp. MCCS 026 was shown. The total fatty acid content of the isolated strain was 25%. The composition of fatty acids in the studied species of microalga was mainly docosanoic acid methyl ester, tetradecanoic acid methyl ester, hexadecanoic acid methyl ester and nonanoic acid methyl ester.

Characterization of hydrocortisone biometabolites and 18S rRNA gene in Chlamydomonas reinhardtii cultures.

A unicellular microalga, Chlamydomonas reinhardtii, was isolated from rice paddy-field soil and water samples and used in the biotransformation of hydrocortisone (1). This strain has not been previously tested for steroid bioconversion. Fermentation was carried out in BG-11 medium supplemented with 0.05% substrate at 25 degrees C for 14 days of incubation. The products obtained were chromatographically purified and characterized using spectroscopic methods. 11b,17 beta-Dihydroxyandrost-4-en-3-one (2), 11 beta-hydroxyandrost-4-en-3,17-dione (3), 11 beta,17 alpha,20 beta,21-tetrahydroxypregn-4-en-3-one (4) and prednisolone (5) were the main products of the bioconversion. The observed bioreaction features were the side chain degradation of the substrate to give compounds 2 and 3 and the 20-ketone reduction and 1,2-dehydrogenation affording compounds 4 and 5, respectively. A time course study showed the accumulation of product 2 from the second day of the fermentation and of compounds 3, 4 and 5 from the third day. All the metabolites reached their maximum concentration in seven days. Microalgal 18S rRNA gene was also amplified by PCR. PCR products were sequenced to confirm their authenticity as 18S rRNA gene of microalgae. The result of PCR blasted with other sequenced microalgae in NCBI showed 100% homology to the 18S small subunit rRNA of two Chlamydomonas reinhardtii spp.

Nucleophilic substitution reactions of pyranose polytosylates.

The 2,3,4-tri-toluenesulfonate ester derivatives of the methyl pyranosides of l-arabinose, d-ribose, d-lyxose, and d-xylose have been prepared, and their substitution reactions with various nucleophiles have been examined. For arabinose, xylose, and ribose, highly regioselective monosubstitutions were observed with benzoate, nitrite, and azide anions. These reactions have led to short and simple routes from d-xylose to l-arabinose derivatives, from l-arabinose to d-xylose derivatives, and from d-ribose to l-lyxose derivatives. The tritosylate derived from methyl alpha-d-lyxopyranoside was unreactive toward nucleophilic substitution reactions, giving instead a dihydropyran product arising from an initial E2 elimination reaction of the 2-tosylate.