RESUMEN
One-pot multicomponent synthesis to assemble compounds has been an efficient method for constructing a compound library. We have developed one-pot tandem copper-catalyzed azidation and CuAAC reactions that afford 1-thiazolyl-1,2,3-triazoles with anticancer activity. By utilizing this one-pot synthetic strategy, we constructed a library of 1-thiazolyl-1,2,3-triazoles in search of the potent lead compound. Furthermore, 1-thiazolyl-1,2,3-triazoles were evaluated for anticancer activity against the multidrug-resistant cancer cells MES-SA/Dx5. Most of the 1-thiazolyl-1,2,3-triazoles revealed cytotoxic effect against cancer cells at micromolar to low micromolar range. Testing some of the most potent compounds (5{4,2-4} and 5{5,1-3}) against the normal cell line Vero showed no significant toxicity (except 5{4,2}) to normal cells. This result indicates that compounds 5{4,3-4} and 5{5,1-3} possessed good potency and selectivity to cancer cells over normal cells.
Asunto(s)
Antineoplásicos/química , Cobre/química , Triazoles/química , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Catálisis , Fluorescencia , Microscopía Electrónica de Rastreo , Triazoles/síntesis química , Triazoles/farmacologíaRESUMEN
BACKGROUND: Intramuscular injection is a popular and effective approach to administer naked plasmid for transgene expression. The use of an adjuvant can provide a straightforward approach for enhancing transgene expression. METHODS: Expression plasmid was formulated with various concentrations of trehalose for injection into the skeletal muscles of C57BL/6 mice. The effects of trehalose on gene dosage and the duration of transgene expression were assessed. The levels of transgene expression were indicated by levels of luciferase expression of the homogenized whole skeletal muscle or by histological X-gal staining of beta-galactosidase expression. Trehalose was also added to serum to examine the ability of protecting the DNA from degradation. RESULTS: It was found that an optimal trehalose concentration of 10 mM will achieve a level of transgene expression that is seven-fold higher than in the absence of trehalose. When compared with other disaccharides, only the incorporation of trehalose can effectively enhance transgene expression. Trehalose is able to improve transgene expression by intramuscular injection at a low gene dosage as well as prolong the duration of transgene expression. CONCLUSIONS: Trehalose is an effective adjuvant for intramuscular administration of naked plasmid with respect to both enhanced levels and prolonged duration of transgene expression, most likely due to retarding plasmid degradation.
Asunto(s)
ADN/administración & dosificación , Expresión Génica , Técnicas de Transferencia de Gen , Plásmidos , Transgenes , Trehalosa/administración & dosificación , Trehalosa/química , Animales , ADN/genética , ADN/metabolismo , Disacáridos/química , Femenino , Dosificación de Gen/efectos de los fármacos , Inyecciones Intramusculares , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Trehalosa/farmacologíaRESUMEN
Using enhancers to improve the transfection efficiency of polyethylenimine (PEI) can circumvent the needs of chemical modifications as well as subsequent purification and characterization of the modified PEI. In this study, we found that incorporating trehalose into the transfection reagent could improve the transgene expression mediated by DNA-PEI complexes. Such enhancements were not observed when trehalose was replaced by other disaccharides. In an effort to explore the mechanisms, we examined how the timing of trehalose treatments and the durations of trehalose affected the percentages of cells expressing green fluorescent protein and the levels of intracellular ethidium monoazide labeled plasmid. Treatments with trehalose for 5-120 min prior to transfection could cause drops in transfection efficiency by 30-50%; such treatments, however, hardly affected the amounts of intracellular plasmid, indicating that the preexistence of intracellular trehalose could reduce transfection efficiency without lowering the endocytic activity. The transfection efficiency remained almost unchanged when the transfected cells were treated with trehalose after the removal of transfection reagents, indicating that trehalose had minimal effects on the machinery of protein synthesis. Despite the enhanced transgene expression, the presence of trehalose during transfection showed inhibitory effects on the internalization of DNA-PEI complexes. Additionally, the extent of enhancement in transgene expression strongly depended on the duration of trehalose. As the above observations suggested, only during the transfection process when complexes and trehalose coexisted, trehalose became an effective enhancer of transgene expression mediated by DNA-PEI complexes possibly by affecting the mechanisms of intracellular trafficking.
Asunto(s)
ADN/administración & dosificación , Iminas/administración & dosificación , Polietilenos/administración & dosificación , Transfección/métodos , Transgenes , Trehalosa/farmacología , Animales , Células CHO , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , ADN/química , Endocitosis/efectos de los fármacos , Citometría de Flujo , Iminas/química , Plásmidos , Polietilenos/química , Factores de TiempoRESUMEN
BACKGROUND: Lipid-based vectors have been widely applied to in vivo and in vitro gene delivery. Disaccharides can effectively stabilize lipid membranes. This study examined whether disaccharides could enhance the transgene expression mediated by lipid-based vectors. METHODS: Different disaccharides were incorporated into the vectors prepared with DOTAP/protamine/DNA (LPD) or with DNA/cationic liposomes containing DOTAP, DOTAP/Chol, DOTAP/DOPE, or DC-Chol/DOPE. The levels of transgene expression and internalized plasmid of CHO cells were represented by the percentages of GFP-positive cells and the fluorescence intensity of ethidium-monoazide covalently labeled plasmid, respectively. The vectors containing either cellobiose or trehalose were also intravenously injected into mouse tail vein to investigate the potentials of in vivo applications. RESULTS: For enhancing the transgene expression, cellobiose was found to be effective for all the vectors whereas maltose decreased the effectiveness of DOTAP/Chol liposomes and LPD. For the internalization of plasmid, most disaccharides were able to increase the cellular delivery of DOTAP, DOTAP/Chol, and DOTAP/DOPE liposomes, but caused decreases in the cellular entry of DC-Chol/DOPE liposomes. An approximately linear correlation between the internalized plasmid and the transgene expression was observed for all the treatments in this study. When the vectors were administered to mouse by intravenous injection, 10-fold and 3-fold increases in the luciferase expression of lung were observed for DOTAP liposomes containing 330 mM cellobiose and trehalose, respectively. CONCLUSIONS: This study showed that using trehalose and cellobiose with a lipid-based delivery system provides a straightforward approach to effectively enhance both in vitro and in vivo transgene expression.
Asunto(s)
Disacáridos/farmacología , Expresión Génica/fisiología , Técnicas de Transferencia de Gen , Transgenes/fisiología , Animales , Células CHO , Proliferación Celular/efectos de los fármacos , Cricetinae , Cricetulus , Vectores Genéticos , Técnicas In Vitro , Lípidos , Liposomas , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Plásmidos/administración & dosificación , Plásmidos/genética , TransfecciónRESUMEN
Branched polyethylenimine (PEI) is a cationic polymer capable of forming self-assembly complexes with DNA to become a highly efficient agent used in gene delivery. Conjugation through the primary amines of PEI is a most commonly used approach further to enable the targeting delivery or to improve the stability of the DNA-polymer complexes. An understanding of how the conjugation affects the transfection mechanisms can help in the design of efficient polycationic vectors. In order to investigate the effects of conjugation, folate and the dextrans of molecular weight 1500 (dex-1500) and 10 000 (dex-10000) were used to prepare three different types of PEI conjugates: dextran-PEI, folate-PEI, and folate-dextran-PEI, which were subsequently employed to form complexes with DNA. These conjugates were found to cause less cytotoxicity than the unmodified PEI as revealed by the MTT method, and to be able to deliver an approximate amount of ethidium monoazide labeled plasmid into the cells. The efficiencies of green fluorescent protein (GFP) expression mediated by these conjugates, however, were less efficient than those mediated by the unmodified PEI. A titration experiment suggested that conjugation through the primary amines of PEI resulted in the loss of relative buffering capacity, a major factor aiding the release of plasmid from the endosomes, presumably because the conjugated molecules hindered the protonation of the PEI conjugates. When a quantitative relationship between relative buffering capacity and transfection efficiency was examined, a threshold of relative buffering capacity, around 50% of the unmodified PEI, was noted to be required for minimal detection of GFP positive cells. In addition, the cytotoxicity could be also related to the relative buffering capacity in an approximately linear trend. It is thus concluded that the severe loss of relative buffering capacity by conjugation might be attributed to the inefficiency of transgene expression mediated by the dextran-PEI conjugates.
Asunto(s)
ADN/administración & dosificación , Dextranos/química , Portadores de Fármacos/química , Polietileneimina/química , Transgenes/fisiología , Tampones (Química) , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , ADN/farmacocinética , Portadores de Fármacos/farmacocinética , Ácido Fólico/química , Ácido Fólico/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Electrónica de Transmisión , Polietileneimina/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
BACKGROUND: Conjugation through primary amines is one of the most commonly used methods to modify polycationic vectors for gene delivery. A better understanding of the effect of the conjugation on the mechanisms of transgene expression can help design efficient polycationic vectors. METHODS: Dextran with a molecular weight of 1500 was grafted onto polyethylenimine (PEI) to produce various degrees of grafting in an effort to investigate how the conjugation affected the mechanisms of transgene expression. Flow cytometry was employed to quantitate the cellular entry of plasmid and the level of transgene expression, which were measured using ethidium monoazide labeled plasmid and green fluorescent protein (GFP), respectively. The buffering capacity of the grafted PEI was determined by titration, and the integrity of the DNA-polymer complexes were examined by exposure to heparin. RESULTS: Grafting of dextran onto PEI was found to significantly diminish the cytotoxicity, buffering capacity, cellular entry, and the integrity of the DNA-polymer complexes. The reductions enlarged as the degree of grafting increased from 0 to 1.84%; however, at an optimal degree of grafting, the dextran-grafted PEI enhanced the percentages of GFP-positive cells to a level 3 times and 1.3 times of those mediated by unmodified PEI for CHO and MDA-MB-231 cells, respectively. CONCLUSIONS: These results demonstrated that the conjugation of dextran onto the primary amines of PEI inhibited the entry of plasmid across the cell membrane, but the change in the structures of the DNA-polymer complexes was able to promote transgene expression when the degrees of conjugation fell below 0.64%.