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The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.

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Articles by Yadollah Omidi in JoVE

 JoVE General

Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay


JoVE 1191 4/03/2009

1,2, 1,3, 1, 2, 1,3

1Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University (Medical Sciences), 2Gifted and Talented Students Office, Educational Development Center, Tabriz University (Medical Sciences), 3School of Advanced Biomedical Sciences, Tabriz University (Medical Sciences)

The MTT assay is an easy and reproducible colorimetric assay for evaluation of cell viability based on reduction of yellow MTT and production of water insoluble purple formazan. Here, the viability of MCF-7 cells upon treatment of nanogenomedicine has been evaluated.

Other articles by Yadollah Omidi on PubMed

Targeting Caveolae for Vesicular Drug Transport

Journal of Controlled Release : Official Journal of the Controlled Release Society. Feb, 2003  |  Pubmed ID: 12618030

Caveolae are morphologically evident as omega-shaped invaginations of the plasma membrane with a diameter of 50-100 nm. They may also exist in a variety of other forms including flattened domains indistinguishable from the plasma membrane itself. At least in some cell types caveolae undertake transport functions including that of the endocytic and transcytotic movement of macromolecules, and indeed microbes and microbial toxins. Opportunities exist for basic and applied investigators working within the pharmaceutical sciences to exploit caveolae membrane interactions with the aim to develop of novel cellular or transcellular drug delivery strategies. This overview article will provide: pertinent information on the biology of the caveolae membrane system; review the various caveolae isolation methods; highlight some of the literature evidence showing that caveolae are functional with regard to macromolecule transport; discuss the role that caveolae could fulfill in the pulmonary absorption of therapeutic proteins from alveolar airspace to capillary blood following inhalational drug delivery, and finally review some very recent work showing proof-of-principle that caveolae domains can be targeted in a tissue-specific manner with highly selective ligands.

Evaluation of the Immortalised Mouse Brain Capillary Endothelial Cell Line, B.End3, As an in Vitro Blood-brain Barrier Model for Drug Uptake and Transport Studies

Brain Research. Nov, 2003  |  Pubmed ID: 14568334

Well-characterised cell lines represent important tools for the study of endogenous solute or xenobiotic transport. A brain microvascular cell line, b.End3, isolated from mice transformed with the Polyoma virus middle T-antigen is available commercially. Here we report the characterisation of some features of b.End3 of relevance to its use in blood-brain barrier transport investigations. The b.End3 cells displayed a distinctive spindle-like squamous morphology in culture. Clathrin coated pits and numerous uncoated intracellular vesicles were evident within the cells, as was the expression of the vesicle-associated proteins, clathrin, caveolin-1, flotillin and dynamin II. In the presence of C6 astroglial co-culture b.End3 monolayers achieved a maximal transendothelial electrical resistance of 130 Omega cm2, but lacked real discrimination with respect to the permeation of transcellular and paracellular probes, e.g. permeability coefficients (x 10(-6) cm s(-1)) for propranolol of approximately 23 vs. 16 for sucrose. RT-PCR analysis confirmed the presence within the b.End3 cells of mRNA transcripts for the following transporters: GLUT-1; MCT 1 and 2; OAT1; Oatp1; mdr 1a and 1b; MRP 1 and 5; beta-alanine, system L and system y+L amino acid carriers; the nucleoside transporters cNT1 and 2, eNT1 and 2, and the tight junctional elements, ZO-1, JAM, occludin, claudin-1 and -5. The b.End3 cells actively accumulated D-glucose in a sodium-independent manner with characteristics consistant with that of GLUT-1. Functionality for P-glycoprotein efflux was evident as assessed by a rhodamine-123 accumulation and retention assay. The system L LAT1/4F2hc amino acid transporter was examined through uptake of L-leucine and L-phenylalanine and provided Km and Vmax values of approximately 16 microM and 350-480 pmol/mg protein/10 min, respectively; the affinity of transport for these substrates being weaker, approximately threefold, when the b.End3 cells were grown in the presence of C6 astroglial factors. Although the b.End3 cells appear unsuitable for transendothelial permeability assessments they display characteristics that would allow their worthwhile use in studies addressing blood-brain barrier transport mechanisms.

Toxicogenomics of Non-viral Vectors for Gene Therapy: a Microarray Study of Lipofectin- and Oligofectamine-induced Gene Expression Changes in Human Epithelial Cells

Journal of Drug Targeting. Jul, 2003  |  Pubmed ID: 14668052

Of the non-viral vectors, cationic lipid (CL) formulations are the most widely studied for the delivery of genes, antisense oligonucleotides and gene silencing nucleic acids such as small interfering RNAs. However, little is known about the impact of these delivery systems on global gene expression in target cells. In an attempt to study the geno-compatibility of CL formulations in target cells, we have used microarrays to examine the effect of Lipofectin and Oligofectamine on the gene expression profiles of human A431 epithelial cells. Using the manufacturer's recommended CL concentrations routinely used for gene delivery, cDNA microarray expression profiling revealed marked changes in the expression of several genes for both Lipofectin- and Oligofectamine-treated cells. Data from the 200 spot arrays housing 160 different genes indicated that Lipofectin or Oligofectamine treatment of A431 cells resulted in more than 2-fold altered expression of 10 and 27 genes, respectively. The downstream functional consequences of CL-induced gene expression alterations led to an increased tendency of cells to enter early apoptosis as assessed by annexin V-FITC flow cytometry analyses. This effect was greater for Oligofectamine than Lipofectin. Observed gene expression changes were not sufficient to induce any significant DNA damage as assessed by single cell gel electrophoresis (COMET) assay. These data highlight the fact that inadvertent gene expression changes can be induced by the delivery formulation alone and that these may, ultimately, have important safety implications for the use of these non-viral vectors in gene-based therapies. Also, the induced non-target gene changes should be taken into consideration in gene therapy or gene silencing experiments using CL formulations where they may potentially mask or interfere with the desired genotype and/or phenotype end-points.

Evaluation of Generation 2 and 3 Poly(propylenimine) Dendrimers for the Potential Cellular Delivery of Antisense Oligonucleotides Targeting the Epidermal Growth Factor Receptor

Pharmaceutical Research. Mar, 2004  |  Pubmed ID: 15070097

To evaluate low generation, G2 and G3, poly(propylenimine) dendrimers for the potential cellular delivery of antisense oligonucleotides (ODNs) targeting the epidermal growth factor receptor (EGFR) in A431 epidermoid carcinoma cells.

Stereospecific Chemical and Enzymatic Stability of Phosphoramidate Triester Prodrugs of D4T in Vitro

European Journal of Pharmaceutical Sciences : Official Journal of the European Federation for Pharmaceutical Sciences. May, 2004  |  Pubmed ID: 15113580

The phosphoramidate triester prodrug approach is widely used to deliver nucleotide forms of nucleoside analogues into target cells. We investigated the stereoselective stability of a series of prodrugs of the anti-HIV agent 2',3'-didehydro-2',3'-dideoxythymidine (d4T). Chemical stability was evaluated in phosphate buffer at pH values of biological relevance (i.e. pH 2.0, 4.6, 7.4). Enzymatic stability was tested in human plasma, in Caco-2 cell homogenates and monolayers and in rat liver. The compounds were relatively stable to chemical hydrolysis. Between 50 and 70% of unchanged prodrug was recovered after 16h incubation in human plasma, with no stereoselective preference for phosphate diastereoisomers. The p-OMe phenyl derivative, however, was an exception and only 5% of one diastereoisomer was recovered. In Caco-2 cells the stability and stereoselectivity largely depended on the experimental conditions: high enzymatic activity and stereoselectivity was observed in cell homogenates, but not in monolayers. In rat liver S9 fractions the stability profile was similar to that in Caco-2 cells and carboxyl ester cleavage appeared to be the sole mechanism of degradation in both media. The large and unpredictable differences in stereoselective metabolic rate of the pronucleotide series here presented suggest that in vivo circulating levels of intact prodrug could exert profoundly different activity or toxicity due to preferential body distribution of one diastereoisomeric form.

Toxicogenomics of Cationic Lipid-based Vectors for Gene Therapy: Impact of Microarray Technology

Current Drug Delivery. Oct, 2005  |  Pubmed ID: 16305446

Implementation of the high-throughput microarray gene expression profiling technology towards "toxicogenomics" has advanced identification process for safer drugs in the century of 'omics' technology. Applying such technology, in fact, to identify mechanisms for cellular toxicity can provide a means to clarify safety liabilities early in the drug discovery and developments process. The underlying principle in gene therapy is primarily targeting a specific gene (e.g., for silencing). Hence, massive efforts have been devoted to validate the gene-based therapeutics, regardless of toxicogenomics potential of delivery systems. Of the gene delivery systems, viral and non-viral vectors, as two main paradigms, have so far been widely used for delivering of the genome-based therapeutics such as oligonucleotide, small interfering RNA and DNA. However, the use of viral vectors was narrowed due to the safety concerns. Non-viral vectors were utilized as safer alternatives for gene delivery in vitro and ex-vivo; though their success for in vivo gene therapy has been limited due to low efficiency and safety issues. Fundamental principle for gene therapy is to deliver gene-based therapeutics into target cells for specific gene targeting ideally with minimal cellular toxicity. Until now, few works have been conducted about geno-compatibility of delivery systems itself, including cationic lipid-based nanosystems. Inadvertent toxicogenomic impact of gene delivery systems (e.g., cationic lipids) may intrinsically affect the outcome of gene therapy, where often only a single desired genetic change is sought. Further, there exists a possibility that gene changes induced by the lipid delivery system itself could exacerbate, attenuate or even mask the desired effects of the gene-based therapeutics. This review will focus on toxicogenomics impact of the cationic lipid-based formulations for gene therapy.

Expression and Transport Functionality of FcRn Within Rat Alveolar Epithelium: a Study in Primary Cell Culture and in the Isolated Perfused Lung

Pharmaceutical Research. Feb, 2006  |  Pubmed ID: 16382279

The neonatal constant region fragment receptor (FcRn) binds and transports IgG. FcRn expression in the upper tracheobronchial airways of the lung is recognized. In this study, we sought to characterize the functional expression of FcRn within alveolar regions of lung tissue.

Toxicogenomics of Drug Delivery Systems: Exploiting Delivery System-induced Changes in Target Gene Expression to Enhance SiRNA Activity

Journal of Drug Targeting. Jan, 2007  |  Pubmed ID: 17365277

Synthetic siRNAs are typically formulated with drug delivery systems (DDS) that improve cellular uptake for optimal gene silencing activity. Here, we show that two PAMAM dendrimer DDS, differing only in their structural architecture, elicit many different gene expression changes in human cells including opposing effects on the expression of epidermal growth factor receptor (EGFR), a gene targeted for silencing by siRNA. Despite providing similar improvements in siRNA uptake, these two formulations led to a approximately 10-fold variation in anti-EGFR siRNA activity. These data show that gene expression changes induced by DDS, separate from their ability to enhance cell uptake, determine 'apparent' siRNA potency and thus offer the possibility of tailoring delivery system-siRNA combinations for additive or synergistic effects on gene silencing.

Primary Porcine Brain Microvascular Endothelial Cells: Biochemical and Functional Characterisation As a Model for Drug Transport and Targeting

Journal of Drug Targeting. May, 2007  |  Pubmed ID: 17487694

The blood-brain barrier (BBB) remains a significant obstacle to the delivery of therapeutic agents into the central nervous system (CNS). Primary cell cultures of brain capillary endothelial cells represent the closest possible phenotype to the in vivo BBB cell providing a convenient model for the study of transport systems and events that mediate solute delivery to the CNS. In this investigation we have characterized an in vitro primary BBB model from porcine brain microvascular endothelial capillary (PBMVEC) cells after recovery from cryopreservation of upto 12 months and studied their modulation by astrocytes. Co-cultures of PBMVECs with astrocytes (C6 astroglioma) resulted in trans-endothelial electrical resistance of up to approximately 900Omega cm2 and marked discrimination between the para- and trans- cellular markers sucrose and propranolol. Micrographs of confluent monolayers of PBMVECs showed the presence of tight junction complexes and vesicles with the morphological characteristics of either caveolae or clathrin coated pits. Extensive RT-PCR evaluation highlighted the expression of tight junction transcripts, ABC transporters, leptin receptor and select nutrient transporters. Functional studies examined the kinetics of transport of glucose, large neutral amino acids and p-glycoprotein (P-gp). Our findings indicate primary PBMVECs retain many barrier characteristics and transport pathways of the in vivo BBB. Further, primary cells can be stored as frozen stocks which can be thawed and cultured without phenotypic drift many months after isolation. Frozen PBMVECs therefore serve as a robust and convenient in vitro cell culture tool for research programs involving CNS drug delivery and targeting and in studies addressing blood-brain barrier transport mechanisms.

Piroxicam Nanoparticles for Ocular Delivery: Physicochemical Characterization and Implementation in Endotoxin-induced Uveitis

Journal of Drug Targeting. Jul, 2007  |  Pubmed ID: 17613659

To investigate the anti-inflammatory impacts of piroxicam nanosuspension, in the current investigation, piroxicam:Eudragit RS100 nanoformulations were used to control inflammatory symptoms in the rabbits with endotoxin-induced uveitis (EIU). The nanoparticles of piroxicam:Eudragit RS100 was formulated using the solvent evaporation/extraction technique. The morphological and physicochemical characteristics of nanoparticles were studied using particle size analysis, X-ray crystallography, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). Drug release profiles were examined by fitting the data to the most common kinetic models. Selected nanosuspensions were used to assess the anti-inflammatory impacts of piroxicam nanoparticles in the rabbits with EIU. The major symptoms of EIU (i.e. inflammation and leukocytes numbers in the aqueous humor) were examined. All the prepared piroxicam formulations using Eudragit RS100 resulted in a nano-range size particles and displayed spherical smooth morphology with positively charged surface, however, the formulated particles of drug alone using same methodology failed to manifest such characteristics. The Eudragit RS100 containing nanoparticles displayed lower crystallinity than piroxicam with no chemical interactions between the drug and polymer molecules. Kinetically, the release profiles of piroxicam from nanoparticles appeared to fit best with the Weibull model and diffusion was the superior phenomenon. The in vivo examinations revealed that the inflammation can be inhibited by the drug:polymer nanosuspension more significantly than the microsuspension of drug alone in the rabbits with EIU. Upon these findings, we propose that the piroxicam:Eudragit RS100 nanosuspensions may be considered as an improved ocular delivery system for locally inhibition of inflammation.

Inhibition of Endotoxin-induced Uveitis by Methylprednisolone Acetate Nanosuspension in Rabbits

Journal of Ocular Pharmacology and Therapeutics : the Official Journal of the Association for Ocular Pharmacology and Therapeutics. Oct, 2007  |  Pubmed ID: 17900230

In this study, nanoformulations of methylprednisolone acetate (MPA) were formulated by using a copolymer of poly(ethylacrylate, methyl-methacrylate and chlorotrimethyl-ammonioethyl methacrylate) to study their impacts on the inhibition of inflammatory symptoms in rabbits with endotoxin-induced uveitis (EIU).

Characterization and Astrocytic Modulation of System L Transporters in Brain Microvasculature Endothelial Cells

Cell Biochemistry and Function. Apr, 2008  |  Pubmed ID: 18210381

Brain trafficking of amino acids is mainly mediated by amino acids transport machineries of the blood-brain barrier (BBB), where astrocytes play a key maintenance role. However, little is known about astrocytes impacts on such transport systems, in particular system L that consists of large and small neutral amino acids (NAAs) transporters, that is, LAT1/4F2hc and LAT2/4F2hc, respectively. In the current investigation, functionality and expression of system L were studied in the immortalized mouse brain microvascular endothelial b.End3 cells cocultured with astrocytes or treated with astrocyte-conditioned media (ACM). LAT2/4F2hc mediated luminal uptake of L-phenylalanine and L-leucine resulted in significantly decreased affinity of system L in b.End3 cells treated with ACM, while LAT2/4F2hc mediated luminal uptake of L-alanine remained unchanged. Gene expression analysis revealed marked upregulation of LAT1 and 4F2hc, but downregulation of LAT2 in b.End3 cells cultured with ACM. The basal to apical transport of L-phenylalanine and L-alanine appeared to be significantly greater than that of the apical to basal direction in b.End3 cells indicating an efflux functionality of system L. No marked influence was observed for transport of L-phenylalanine in b.End3 cells cocultured with astrocytes, while a slight decrease was seen for L-alanine in the basal to apical direction. Based on our findings, we propose that system L functions as influx and/or efflux transport machinery displaying a greater propensity for the outward transport of large and small NAAs. Astrocytes appeared to modulate the transcriptic expression and uptake functionalities of system L, but not the transport activities.

Kinetic Analysis of Drug Release from Nanoparticles

Journal of Pharmacy & Pharmaceutical Sciences : a Publication of the Canadian Society for Pharmaceutical Sciences, Société Canadienne Des Sciences Pharmaceutiques. 2008  |  Pubmed ID: 18471378

Comparative drug release kinetics from nanoparticles was carried out using conventional and our novel models with the aim of finding a general model applicable to multi mechanistic release. Theoretical justification for the two best general models was also provided for the first time.

Ocular Novel Drug Delivery: Impacts of Membranes and Barriers

Expert Opinion on Drug Delivery. May, 2008  |  Pubmed ID: 18491982

Ocular drug delivery is an extremely challenging area due to its restrictive barrier functionalities.

Microarray Analysis of the Toxicogenomics and the Genotoxic Potential of a Cationic Lipid-based Gene Delivery Nanosystem in Human Alveolar Epithelial A549 Cells

Toxicology Mechanisms and Methods. 2008  |  Pubmed ID: 20020904

ABSTRACT Viral and nonviral vectors have been widely used in gene therapy as delivery reagents for nucleic acids. Toxicity with viral vectors has increasingly led to the search for suitable nonviral vectors, such as cationic lipids/polymers, as potentially safer alternatives. However, little is known about the genomic toxicity of these delivery systems in target cells/tissues. In the current investigation, we report on the toxicogenomics and genotoxicity of cationic lipid Oligofectamine (OF) nanosystems in human alveolar epithelial A549 cells. To investigate the nature and the ontology of the gene expression changes in A549 cells upon treatment with OF nanoliposomes, microarray gene expression profiling methodology was utilized. For microarray analysis, cyanine (Cy3/Cy5)-labeled cDNA samples from treated and untreated cells were hybridized on target arrays housing 200 genes. Both OF and OF-DNA lipoplex induced significant gene expression changes belonging to the different genomic ontologies such as cell defense and apoptosis pathways. Flow cytometry analyses revealed induction of apoptosis in A549 cells treated with these nanosystems that is likely due to interactions and/or deterioration of the cell membranes. However, no DNA damage was detected by the Comet assay. These data suggest that cationic nanoliposomes in the absence of direct DNA damage elicit multiple gene expression changes in A549 cells that may compromise the main goals of gene medicine where only therapy-defined gene changes are required.

Frequency of Five Important CYP2D6 Alleles Within an Iranian Population (Eastern Azerbaijan)

Genetic Testing and Molecular Biomarkers. Oct, 2009  |  Pubmed ID: 19715474

Polymorphisms in cytochrome P450 genes encoding enzymes of critical importance for drug metabolism have the highest genetic influence on interindividual variations in drug bioavailability. Human CYP2D6 enzyme is claimed to be polymorphically expressed among different ethnic groups. It has been suggested to account for a large part of the interindividual differences in drug metabolism and pharmacokinetics. In the current investigation, 100 healthy unrelated subjects living in Tabriz, Iran, were randomly selected. Genotyping was designed to determine the frequencies of five major and important alleles: CYP2D6*2, CYP2D6*4, CYP2D6*5, CYP2D6*10, and CYP2D6*17. After collecting venous blood samples, polymerase chain reaction-restriction fragment length polymorphism methodology was performed for detection of the alleles (except CYP2D6*5, which has been detected using an allele-specific polymerase chain reaction procedure). Finally, the obtained data were used to determine the allele frequencies. The frequencies for CYP2D6 alleles *2, *4, *5, and *10 were 32%, 12.5%, 3%, and 9%, respectively. CYP2D6*17 was completely absent in this study group. Poor metabolizer phenotype can be related to *4/*4 and *4/*5 genotypes with a total frequency of 4%. This is the first study of the CYP2D6 genetic polymorphism in an Iranian population. The frequencies of the studied alleles resulted in degrees of differences between this population and Orientals, Saudi Arabians, and Caucasians, while similarities to the reported results obtained from the studies among Mediterraneans and South Indians are noticeable.

Target Therapy of Cancer: Implementation of Monoclonal Antibodies and Nanobodies

Human Antibodies. 2009  |  Pubmed ID: 19729803

In the past decades, the mainstay of systemic therapy for solid and haematological malignancies was chemotherapy; nevertheless this modality has the drawbacks such as drug resistance and eliciting sever cytotoxicity in the normal tissue. To resolve such downsides, the cancer therapy modalities need to be advanced with more effective and tolerable treatments to specifically target the malignant cell with minimal adverse consequences. In fact, characteristically, the malignant diseases are self sufficiency in growth signals along with insensitivity to growth inhibition. They can also evade from apoptosis, have limitless replicative potential, induce angiogenesis and possess metastasis potential. Given that the most of these characteristics are often due to genetic defects, thus key to the development of targeted therapies is the ability to use such processes to phenotypically distinguish the tumor from its normal counterpart by its specific/selective markers. The therapeutic monoclonal antibodies (mAbs) are deemed to be a class of novel agents that can specifically target and disrupt molecular pathways underlying tumorigenesis. The mAbs are produced by a single clone of B-cells, and are monospecific and homogeneous. Since Kohler and Milstein heralded a new era in antibody research and clinical development by the discovery of hybridoma technology in 1975, more than 20 mAbs have been approved by the US Food and Drug Administration (FDA) for treatment of obdurate diseases, including different types of cancers. Mouse hybridomas were the first reliable source of monoclonal antibodies which were developed for several in vivo therapeutic applications. Accordingly, the recombinant antibodies have been reduced in size, rebuilt into multivalent molecules and fused with different moieties such as radionuclides, toxins and enzymes. The emergence of recombinant technologies, transgenic animals and phage display technology has revolutionized the selection, humanization and production of antibodies. This review focuses on implementation of the mAbs and nanobodies fragments for cancer therapy.

Prevention of Selenite-induced Cataractogenesis in Wistar Albino Rats by Aqueous Extract of Garlic

Journal of Ocular Pharmacology and Therapeutics : the Official Journal of the Association for Ocular Pharmacology and Therapeutics. Oct, 2009  |  Pubmed ID: 19857100

The main aim of this study was to evaluate the inhibitory impacts of the aqueous extract of garlic (Ga) on the formation of cataract induced by sodium selenite (Se).

Application of Quartz Crystal Nanobalance for Simultaneous Determination of Vanillylmandelic and Homovanillic Acids by a Net Analyte Signal-based Method

Applied Biochemistry and Biotechnology. Oct, 2009  |  Pubmed ID: 19089651

Homovanillic acid (HVA) and vanillylmandelic acid (VMA) were selectively determined by quartz crystal nanobalance sensor in conjunction with net analyte signal (NAS)-based method called HLA/GO. An orthogonal design was applied for the formation of calibration and prediction sets including HVA, VMA, and some common and structurally similar urine compounds. The selection of the optimal time range involved the calculation of the NAS regression plot in any considered time window for each test sample. The searching of a region with maximum linearity of NAS regression plot (minimum error indicator) and minimum of predicted error sum of squares value was carried out by applying a moving window strategy. Based on the obtained results, the differences on the adsorption profiles in the time range between 1 and 300 s were used to determine mixtures of compounds by HLA/GO method. Several figures of merit like selectivity, sensitivity, analytical sensitivity, and limit of detection were calculated for both compounds. The results showed that the method was successfully applied for the determination of VMA and HVA.

Development and Characterization of Monoclonal Antibodies Against Human Epidermal Growth Factor Receptor in Balb/c Mice

Human Antibodies. 2009  |  Pubmed ID: 19478394

For production of monoclonal antibody (mAb) against human epidermal growth factor receptor (EGFR), first, five female Balb/c mice 6 to 8 weeks old were immunized against A431 tumoral cells that express more EGFR on its membrane in four periods and the most immune mouse was selected for fusion. The fusion of mouse's spleen immune cells with SP2/0 cells (myeloma cells) were fulfilled in presence of Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells were screened for detection of antibody by ELISA. The suitable clones were selected for limiting dilution (L.D). Large scale of monoclonal antibodies was produced in vitro and ascetic fluid. In this investigation, 280 clones were obtained, 27 of which displayed absorbance more than 1. Of these, 3 clones represented absorbance about 1.7 and selected for limiting dilution. The yield of limiting dilution was 8 monoclones with absorbance about 2. These results indicate that such monoclonal antibodies against EGFR can be used in diagnosis and treatment of tumors with membranous EGFR.

Induction of Human Alveolar Epithelial Cell Growth Factor Receptors by Dendrimeric Nanostructures

International Journal of Toxicology. Mar-Apr, 2009  |  Pubmed ID: 19482835

Although nonviral dendrimeric nanostructures have been widely used as gene delivery systems, key questions about target cells responses to these nanostructures are yet to be answered. Here, we report the responsiveness of A431 and A549 cells upon treatment with polypropylenimine diaminobutane (DAB) dendrimers nanosystems. Complexation of DAB dendrimers with DNA reduced the zeta potential of nanostructures, but increased their size. Fluorescence microscopy revealed high transfection efficiency in both cell lines treated with DAB dendrimers with induced cytotoxicity evidenced by MTT assay. The A549 cells showed upregulation of epidermal growth factor receptor (EGFR) and its downstream signalling biomolecule Akt kinase upon treatment with DAB dendrimers, while no changes were observed in A431 cells. Based on our findings, the biological impacts of these nanosystems appeared to be cell dependent. Thus, the biological responses of target cells should be taken into account when these nanostructures are used as gene delivery system.

Bioimpacts of Anti Epidermal Growth Factor Receptor Antisense Complexed with Polyamidoamine Dendrimers in Human Lung Epithelial Adenocarcinoma Cells

Journal of Biomedical Nanotechnology. Aug, 2010  |  Pubmed ID: 21323109

Lung cancer is still one of the leading causes of malignancy related deaths worldwide despite recent advances in diagnosis and therapy. Among various biomarkers detected in cancerous cells, the epidermal growth factor receptor (EGFR) plays a key role in initiation/promotion of several malignancies. Thus, a number of studies have been carried out to target this important receptor. In the present study, effects of anti-EGFR antisense (AS-ODN) nanoparticles formulated with star burst polyamidoamine (PAMAM) dendrimers on the expression of EGFR and its downstream molecules were investigated in human lung cancer A549 cells. Complexation of dendrimers with AS-ODN reduced the zeta potential of nanostructures (approximately 10 mV), but increased their size (approximately150 nm). Fluorescence microscopy revealed high transfection efficiency which was further confirmed with flow cytometry technique. Significant cell growth reduction in the treated cells was detected using MTT assay and marked downregulation of EGFR and some of its downstream signaling biomolecule (i.e., Akt kinase) were observed. Microarray profile revealed nonspecific changes in gene expression in A549 cells upon treatment with PAMAM dendrimers alone or as complexed with As-ODN, while comet assay showed no DNA damage. Based on our findings, EGFR targeting antisense is able to inhibit the growth of A549 cells via downregulation of EGFR and Akt kinase, nevertheless these nanopolyplexes can also induce nonspecific bioimpacts in target cells.

Rapid and Simple Methodology for Isolation of High Quality Genomic DNA from Coniferous Tissues (Taxus Baccata)

Molecular Biology Reports. Feb, 2010  |  Pubmed ID: 19649730

Various investigations have been so far performed for extraction of genomic DNA from plant tissues, in which the extracted intact DNA can be exploited for a diverse range of biological studies. Extraction of high quality DNA from leathery plant tissues (e.g., coniferous organs) appears to be a critical stage. Moreover, for some species such as Taxus trees, bioprocess engineering and biosynthesis of secondary metabolites (e.g., paclitaxel) is a crucial step due to the restrictions associated with extinction of these species. However, extraction of intact genomic DNA from these plants still demands a rapid, easy and efficient protocol. To pursue such aim, in the current work, we report on the development of a simple and highly efficient method for the extraction of DNA from Taxus baccata. Based upon our protocol, interfering phenolic compounds were removed from extraction using polyvinylpyrrolidone and RNA contamination was resolved using LiCl. By employing this method, high quality genomic DNA was successfully extracted from leaves of T. baccata. The quality of extracted DNA was validated by various techniques such as RAPD marker, restriction digestions and pre-AFLP. Upon our findings, we propose this simple method to be considered for extraction of DNA from leathery plant tissues.

Barrier Functionality and Transport Machineries of Human ECV304 Cells

Medical Science Monitor : International Medical Journal of Experimental and Clinical Research. Jan, 2010  |  Pubmed ID: 20037486

The lack of a brain-derived endothelial cell-based model has led researchers to exploit non-brain endothelial/epithelial cells as in vitro models for blood-brain barrier (BBB) investigations. Of these, the endothelial-like human ECV304 cell line was initially characterized as endothelial cells and has been widely used as an in vitro BBB model. It was later shown to be T24 bladder carcinoma epithelial cells. To pursue its potential as a cell-based model for drug screening and transport machineries, ECV304 cells were evaluated for their barrier and plasma membrane characteristics.

Synthesis and in Vitro Release of Adriamycin from Star-shaped Poly(lactide-co-glycolide) Nano- and Microparticles

Journal of Pharmaceutical Sciences. Aug, 2010  |  Pubmed ID: 20229603

The star-shaped poly(lactide-co-glycolide (PLGA)-beta-cyclodextrin (PLGA-beta-CD) copolymer was synthesized by reacting L-lactide, glycolide, and beta-cyclodextrin in the presence of stannous octoate as a catalyst. The structure of PLGA-beta-CD copolymer was confirmed with (1)H-NMR, (13)C-NMR, and FT-IR spectra. Adriamycin (ADR), which is an antitumor antibiotic, was encapsulated within micro- and nanoparticles made of PLGA-beta-CD with a modified double emulsion method. Relatively low amount of beta-CD and catalyst were used in order to obtain high molecular weight polymers. Differential scanning calorimetry (DSC) was used to determine the thermal properties of star-shaped copolymers. The reduction of interactions between the star-shaped polyester molecules is due to their branched structure lowered T(g) and T(m) compared to linear PLGA copolymers. Effects of the experimental parameters, such as copolymer composition, ADR concentration, copolymer concentration, and poly(vinyl alcohol) concentration, on particular size and encapsulation efficiency were investigated. An increase in the internal aqueous phase volume led to a decrease in particles average size. A decrease in the polymer concentration resulted in increasing the particle average size from 135.5 to 325.6 nm. The high entrapment efficiency (EE) (about 65%) was obtained for 220 microm particles. All of the release profiles indicated a close relationship between each formulation variable and the amount of ADR released.

Physicochemical and Biological Properties of Self-assembled Antisense/poly(amidoamine) Dendrimer Nanoparticles: the Effect of Dendrimer Generation and Charge Ratio

International Journal of Nanomedicine. 2010  |  Pubmed ID: 20517481

To gain a deeper understanding of the physicochemical phenomenon of self-assembled nanoparticles of different generations and ratios of poly (amidoamine) dendrimer (PAMAM) dendrimer and a short-stranded DNA (antisense oligonucleotide), multiple methods were used to characterize these nanoparticles including photon correlation spectroscopy (PCS); zeta potential measurement; and atomic force microscopy (AFM). PCS and AFM results revealed that, in contrast to larger molecules of DNA, smaller molecules produce more heterodisperse and large nanoparticles when they are condensed with a cationic dendrimer. AFM images also showed that such nanoparticles were spherical. The stability of the antisense content of the nanoparticles was investigated over different charge ratios using polyacrylamide gel electrophoresis. It was clear from such analyses that much more than charge neutrality point was required to obtain stable nanoparticles. For cell uptake, self-assembled nanoparticles were prepared with PAMAM G5 and 5'-FITC labeled antisense and the uptake experiment was carried out in T47D cell culture. This investigation also shows that the cytotoxicity of the nanoparticles was dependent upon the generation and charge ratio of the PAMAM dendrimer, and the antisense concentration had no significant effect on the cytotoxicity.

Impacts of Anti-EGFR Monoclonal Antibody in Prostate Cancer PC3 Cells

Human Antibodies. 2010  |  Pubmed ID: 20826931

Epidermal growth factor receptor (EGFR) network appears to be a rich target for prostate cancer. Thus, in this current investigation, to pursue our newly developed anti-EGFR monoclonal antibody (mAb) in prostate cancer, its inhibitory effect was investigated in PC3 cells. The binding specificity of antibody was examined by flow cytometry and immunofluorescence staining. The cultivated cells were treated with various doses of the anti-EGFR mAb at different time points and the cellular and/or molecular impacts were assessed. MTT assay was utilized to examine the cytotoxic effects. Semi-quantitative RT-PCR was used to evaluate the expression of EGFR and some important apoptosis signaling molecules (e.g., MAPK-1, STAT-5, Akt-1 kinase). Flow cytometric and immunofluorescence staining analyses showed that the anti-EGFR mAb can bind to EGFR with high specificity. The results revealed that the anti-EGFR mAb can inhibit cell growth in a dose and time dependent manner. RT-PCR analysis revealed that the binding of anti-EGFR mAb to its receptors can eventually result in downregulation of Akt-1 kinase gene, but not MAPK-1, STAT-5 and EGFR genes. Based on our findings, it can be concluded that Akt-1 is the most important downstream signaling molecules affected by anti-EGFR mAbs.

Synthesis and in Vitro Studies of Cross-linked Hydrogel Nanoparticles Containing Amoxicillin

Journal of Pharmaceutical Sciences. Nov, 2010  |  Pubmed ID: 21053368

In this paper, we report the synthesis and characterization of a novel cross-linked N-isopropylacrylamide-acrylic acid-hydroxyethyl methacrylate [P (NIPASM-AA-HEM)] hydrogel nanoparticles (NPs) containing amoxicillin. The aim of present study was to investigate whether these hydrogel NPs have the potential to be used in antibiotic delivery to stomach for treatment of Helicobacter pylori. Amoxicillin-loaded hydrogel NPs were prepared using cross-linked P (NIPASM-AA-HEM) as mucoadhesive polymer for the potential use of treating gastric and duodenal ulcers. Aiming at predicting the in vivo behavior of the amoxicillin-loaded NPs, the physicochemical properties in terms of entrapment efficiency (EE%), mean diameter, and morphology of NPs was evaluated. The dependence of the EE% of the drug on the organic to aqueous phase ratio was also studied. The profile of amoxicillin release from P (NIPASM-AA-HEM) NPs system was studied under various conditions. In all these experiments, amoxicillin release in the free form was studied by ultraviolet (UV) spectrophotometric analysis. Experimental results showed that at pH 7.4, drug release rises when polymer concentration in the formulation increases; in human plasma on the contrary, drug release is reduced as concentration of the polymer in the formulation rises. In vitro amoxicillin release rate was also higher in pH 1 than that in pH 7.4. About 88.5% of amoxicillin entrapped in the NPs was released in 4 h in the pH 1.0 medium, whereas in phosphate buffer at pH 7.4 no more than 45% was released after 4 h incubation at 37°C. Amoxicillin concentration in rat's gastric tissue was determined. The results of in vivo studies showed that the hydrogel NPs enhance drug concentration at topical site than powder amoxicillin. Thus, amoxicillin-loaded hydrogel NPs may provide therapeutic concentration at a much lower dose that may reduce the adverse effects of amoxicillin in high doses. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci.

A Robust Universal Method for Extraction of Genomic DNA from Bacterial Species

Mikrobiologiia. Jul-Aug, 2010  |  Pubmed ID: 21058509

The intactness of DNA is the keystone of genome-based clinical investigations, where rapid molecular detection of life-threatening bacteria is largely dependent on the isolation of high-quality DNA. Various protocols have been so far developed for genomic DNA isolation from bacteria, most of which have been claimed to be reproducible with relatively good yields of high-quality DNA. Nonetheless, they are not fully applicable to various types of bacteria, their processing cost is relatively high, and some toxic reagents are used. The routine protocols for DNA extraction appear to be sensitive to species diversity, and may fail to produce high-quality DNA from different species. Such protocols remain time-consuming and tedious, thus to resolve some of these impediments, we report development of a very simple, rapid, and high-throughput protocol for extracting of high-quality DNA from different bacterial species. Based upon our protocol, interfering phenolic compounds were removed from extraction using polyvinylpyrrolidone (PVP) and RNA contamination was precipitated using LiCI. The UV spectrophotometric and gel electrophoresis analysis resulted in high A260/A280 ratio (>1.8) with high intactness of DNA. Subsequent evaluations were performed using some quality-dependent techniques (e.g., RAPD marker and restriction digestions). The isolated DNA from 9 different bacterial species confirmed the accuracy of this protocol which requires no enzymatic processing and accordingly its low-cost making it an appropriate method f r large-scale DNA isolation fromvarious bacterial species.

Synthesis and in Vitro Studies of Cross-linked Hydrogel Nanoparticles Containing Amoxicillin

Journal of Pharmaceutical Sciences. Mar, 2011  |  Pubmed ID: 21280053

In this paper, we report the synthesis and characterization of a novel cross-linked N-isopropylacrylamide-acrylic acid-hydroxyethyl methacrylate [P (NIPASM-AA-HEM)] hydrogel nanoparticles (NPs) containing amoxicillin. The aim of present study was to investigate whether these hydrogel NPs have the potential to be used in antibiotic delivery to stomach for treatment of Helicobacter pylori. Amoxicillin-loaded hydrogel NPs were prepared using cross-linked P (NIPASM-AA-HEM) as mucoadhesive polymer for the potential use of treating gastric and duodenal ulcers. Aiming at predicting the in vivo behavior of the amoxicillin-loaded NPs, the physicochemical properties in terms of entrapment efficiency (EE%), mean diameter, and morphology of NPs was evaluated. The dependence of the EE% of the drug on the organic to aqueous phase ratio was also studied. The profile of amoxicillin release from P (NIPASM-AA-HEM) NPs system was studied under various conditions. In all these experiments, amoxicillin release in the free form was studied by ultraviolet (UV) spectrophotometric analysis. Experimental results showed that at pH 7.4, drug release rises when polymer concentration in the formulation increases; in human plasma on the contrary, drug release is reduced as concentration of the polymer in the formulation rises. In vitro amoxicillin release rate was also higher in pH 1 than that in pH 7.4. About 88.5% of amoxicillin entrapped in the NPs was released in 4 h in the pH 1.0 medium, whereas in phosphate buffer at pH 7.4 no more than 45% was released after 4 h incubation at 37 °C. Amoxicillin concentration in rat's gastric tissue was determined. The results of in vivo studies showed that the hydrogel NPs enhance drug concentration at topical site than powder amoxicillin. Thus, amoxicillin-loaded hydrogel NPs may provide therapeutic concentration at a much lower dose that may reduce the adverse effects of amoxicillin in high doses.

Molecular Considerations for Development of Phage Antibody Libraries

Journal of Drug Targeting. Sep, 2011  |  Pubmed ID: 21950316

Nowadays, phage display libraries are used as robust tools for discovery and evolution of peptide/protein based drugs as well as targeting molecules, in particular monoclonal antibodies (mAbs) and its fragments (i.e., scFvs, Fabs, or bivalent F(ab')(2)). Phage display technology, as a molecular diversity approach, enables selection of antibody fragments (e.g., scFv/Fab) with high affinity, specificity and effector functions against various targets. However, such selection process itself is largely dependent upon various molecular factors such as methods for construction of phage library, phage/phagemid vectors, helper phage, host cells and biopanning processes. The current review article provides important molecular considerations for successful development of phage antibody libraries that may be used as a platform for translation of antibody fragments into viable diagnostic/therapeutic reagents.

Chemical Variation of the Essential Oil of Prangos Uloptera DC. at Different Stages of Growth

Natural Product Research. Apr, 2011  |  Pubmed ID: 19606386

In the current investigation, the influence of the plant growth stages (prior to emergence of flower heads or vegetative, anthesis and fruiting) on the essential oil content and composition in the aerial parts of Prangos uloptera DC. was studied. Quantitative and qualitative differences were found among oil of aerial parts at three phenological stages. The principle compounds of the oil before emergence of flower heads were saferole (21.6%) and α-pinene (20%), while the oil at anthesis was composed of α-bisabolol (30.5%), saferole (19.11%) and (+)-spathulenol (12.9%). The oil at fruiting stage displayed γ-terpinene (35.5%) and trans-anethole (23.5%) as major constituents. These findings may confer valuable information regarding chemical composition of P. uloptera DC., which could be beneficial in the fields of herbal medicine.

Novel Water-soluble Polyurethane Nanomicelles for Cancer Chemotherapy: Physicochemical Characterization and Cellular Activities

Journal of Nanobiotechnology. Jan, 2012  |  Pubmed ID: 22221539

ABSTRACT: BACKGROUND: Efficient delivery of anticancer chemotherapies such as paclitaxel (PTX) can improve treatment strategy in a variety of tumors such as breast and ovarian cancers. Accordingly, researches on polymeric nanomicelles continue to find suitable delivery systems. However, due to biocompatibility concerns, a few micellar nanoformulations have exquisitely been translated into clinical uses. Here, we report the synthesis of novel water-soluble nanomicelles using bioactive polyurethane (PU) polymer and efficient delivery of PTX in the human breast cancer MCF-7 cells. RESULTS: The amphiphilic polyurethane was prepared through formation of urethane bounds between hydroxyl groups in poly(tetramethylene ether) glycol (PTMEG) and dimethylol propionic acid with isocyanate groups in toluene diisocyanate (TDI). The free isocyanate groups were blocked with phenol, while the free carboxyl groups of dimethylol propionic acid were reacted with triethylamine to attain ionic centers in the polymer backbone. These hydrophobic PTMEG blocks displayed self-assembly forming polymeric nanomicelles in water. The PTX loaded PU nanomicelles showed suitable physical stability, negative zeta potential charge (-43) and high loading efficiency (80%) with low level of critical micelle concentration (CMC). In vitro drug release profile showed a faster rate of drug liberation at pH 5.4 as compared to that of pH 7.4, implying involvement of a pH-sensitive mechanism for drug release from the nanomicelles. The kinetic of release exquisitely obeyed the Higuchi model, confirming involvement of diffusion and somewhat erosion at pH 5.4. These nanomicelles significantly inhibited the growth and proliferation of the human breast cancer MCF-7 cells, leading them to apoptosis. The real time RT-PCR analysis confirmed the activation of apoptosis as result of liberation of cytochrome c in the cells treated with the PTX loaded PU nanomicelles. The comet assay analysis showed somewhat DNA fragmentation in the treated cells. CONCLUSIONS: Based upon these findings, we propose that the bioactive waterborne polyurethane nanomicelles can be used as an effective nanocarrier for delivery of anticancer chemotherapies such as paclitaxel.

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