Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based medicine and/or its carrier may elicit cellular toxicity. For assessment of such cytotoxicity, a common methodology is largely dependent upon utilization of the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay which has been widely used as a colorimetric approach based on the activity of mitochondrial dehydrogenase enzymes in cells. In this current investigation, MCF-7 cells were inoculated in 96-well plate and at 50% confluency they were treated with different nanopolyplexes and subjected to MTT assay after 24 hours. Water soluble yellow MTT is metabolized by the metabolically active cells to the water insoluble purple formazan, which is further dissolved in dimethylsulfoxide and Sornson s buffer pH 10.5. The resultant product can be quantified by spectrophotometry using a plate reader at 570 nm. Protocol MCF-7 seeding in 96-well plate:MCF-7 cells were cultured in 25 t-flask in medium containing Dulbecco's Modified Eagle's Medium (DMEM), 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO2, 95% air and complete humidity. Once reached~90% confluency, they were detached using 0.05% trypsin/EDTA and counted by means of trypan blue and hemocytometer. These cells were then resuspended at a concentration of 4×10 4 cells/cm 2 and added onto 96-well plate (i.e., 250 μl/well) by an 8-channel pipette. For background absorption, some wells were remained cell-free, i.e. as blank control. Treating cells with different nanopolyplexes:At 40-50% confluency (48 hours post seeding), the cultivated cells were treated with nanostructured starburst polyamidoamine dendrimers (i.e., Superfect® and Polyfect®) and a novel test polymer following the transfection instruction provided by supplier. Cells were also treated with EGFR and scrambled antisense alone, and with the three different nanopolyplexes of these two oligonucleotides and with polymers (n=4). Four wells were remained untreated as control. After 4 hours the treatment media were removed and replenished with fresh media. MTT assay for evaluating cell viability:MTT assay was performed 24 hours after transfection. For this purpose, MTT solution was prepared at 1mg/ml in PBS and was filtered through a 0.2 µm filter. Then, 50 µl of MTT plus 200 µl of DMEM without phenol red were added into each well, except the cell-free blank wells. Cells were incubated for 4 hours at 37°C with 5% CO2, 95% air and complete humidity. After 4 hours, the MTT solution was removed and replaced with 200 µl of DMSO and 25µl Sorenson's glycine buffer (glycine 0.1M, NaCl 0.1M, pH:10.5 with 0.1 NaOH). The plate was further incubated for 5 min at room temperature, and the optical density (OD) of the wells was determined using a plate reader at a test wavelength of 570 nm and a reference wavelength of 630 nm. DiscussionThe MTT assay is deemed to be a versatile method and accordingly the viabilit...
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.
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.
The solubilities of phenothiazine in water, ethanol, and propylene glycol were measured at (298.2 to 338.2) K. Also, the solubility of phenothiazine in binary mixtures of ethanol þ water, propylene glycol þ water, and ethanol þ propylene glycol, and the ternary mixture of ethanol þ propylene glycol þ water was investigated. The van't Hoff equation was used to correlate the solubility of phenothiazine in monosolvents at different temperatures. The solubility values of phenothiazine in binary and ternary mixtures of solvents were calculated using the JouybanÀAcree model (Jouyban, A.; Acree, W. E., Jr. J. Chem. Eng. Data 2009, 54, 1168À1170). The mean deviation was used as an error criterion. The overall mean deviation of correlated solubility data in monosolvents at different temperatures and in mixed solvents at 298.2 K were 2.8 % and 14.2 %, respectively. ' INTRODUCTIONSolubility is an important physicochemical property which plays basic role in most pharmaceutical and industrial processes. To investigate this property different tools have been used such as experimental techniques, mathematical calculations, and simulation. Usually the low solubility of pharmaceutical compounds causes them to fail during the drug development process. Different factors influence the solubility in a medium, some of which include cosolvents, temperature, pH of the solution, and presence of surfactants.Phenothiazine, a triheterocyclic compound (see Figure 1 for its structure), is a veterinary antihelminthic drug, and its derivatives are widely used in pharmacotherapy. Phenothiazine is one of the oldest lead compounds in medicinal chemistry, synthesized in 1883, and clinical applications of the generated drugs from this lead compound were reported in 1891 as antimalaria drug, in 1930s as antifungal, in 1940s as antihelmentic, in 1947 as antihistaminic, in 1951 as antipsychotic, in 1990 as antioxidant, and in 2009 as a promising drug in Alzheimer disease. 1 Solubility data of phenothiazine could be valuable in pharmaceutical applications as many phenothiazine derivatives are of the main and important pharmaceutical compounds. Hoover et al. 2 previously presented mathematical correlation of phenothiazine solubilities in organic solvents with the Abraham solvation parameter model following experimental determination of this solute in monosolvents at 298.2 K. 2 To the best of our knowledge, these are the only reported solubility data for phenothiazine in the literature. Thermodynamic parameters, solubilities, and interactions with micelles of a number of phenothiazine derivative drugs were investigated by Mandal et al. 3À5 The aims of this study are to determine the solubility of phenothiazine in water, ethanol, and propylene glycol at (298.2 to 338.2) K and in ethanol þ water, propylene glycol þ water, ethanol þ propylene glycol, and ethanol þ propylene glycol þ water mixtures at 298.2 K. In addition, the solubility correlations of phenothiazine in the monosolvents at different temperatures and their mixtures are investigate...
Solubility of 2-hydroxybenzoic acid (salicylic acid, with measured melting point of 432 K) in water, 1-propanol, 2-propanol, and 2-propanone was determined at (298.2 to 338.2) K and atmospheric pressure. Also, the solubility of salicylic acid in binary mixtures of 1-propanol (1) + water (2), 2-propanol (1) + water (2), and 2-propanone (1) + water (2) at 298.2 K and atmospheric pressure was investigated. Phase separation occurred in x 1 = 0.114, 0.167 of 1-propanol (1) + water (2) and x 1 = 0.171, 0.237 of 2-propanone (1) + water (2) mixtures. The van't Hoff and Grant equations were used to correlate the solubility of salicylic acid in monosolvents at different temperatures. The solubility values of salicylic acid in binary mixtures of solvents were calculated using the Jouyban−Acree model (J. Chem. Eng. Data 2009Data , 54, 1168Data −1170. The mean deviation was used as an error criterion. The overall mean deviation of correlated solubility data in monosolvents at different temperatures, and in mixed solvents at 298.2 K were 1.0 % and 8.3 %, respectively. ■ INTRODUCTION2-Hydroxybenzoic or salicylic acid, a phenolic acid compound, is a metabolite of salicin, one of the most ancient pain relievers extracted from the bark of willow (Figure 1). Salicylic acid, also an active metabolite of acetylsalicylic acid, shows antiinflammatory effects and is used in topical formulations as a chemical exfoliating agent. It is used in the organic synthesis of many other medicinal compounds including salicylates (its ester and salt derivatives) and acetylsalicylic acid (Aspirin). In recent years, it has been used in cocrystal synthesis of medicinal compounds with a desirable increase in solubility properties. Hence, solubility data of this compound might be of interest for chemists and formulator researchers in chemical, pharmaceutical, food, and cosmetic industries.There are a number of previously reported solubility data for salicylic acid in monosolvents and mixtures of solvents at different temperatures. Solubility data for salicylic acid in monosolvents at different temperatures were reported for water, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl-2-propanol, 1-pentanol, 1-octanol, dibutyl ether, ethylene glycol, propylene glycol, glycerin, acetone, ethyl acetate, butyl acetate, acetic acid, ethyl cellosolve, 1,4-dioxane, tetrahydrofuran, 2-propanone, 2-butanone, cyclohexanone, cyclohexanol, acetonitrile, chloroform, carbon tetrachloride, benzene, benzyl alcohol, and xylene.1−7 Salicylic acid solubility data in mixtures of solvents were reported for water and either methanol, ethanol, 1-propanol, 2-propanone, 2-ethoxyethanol, 1,4-dioxane, or polyethylene glycol 300; methanol and either benzene, 1,4-dioxane, chloroform, or ethyl acetate; ethanol and either benzene, 1,4-dioxane, chloroform, or ethyl acetate; 1-propanol and either benzene, 1,4-dioxane, chloroform, or ethyl acetate; 1-butanol and either benzene, 1,4-dioxane, chloroform, or ethyl acetate; ethylene glyco...
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