RESUMEN
PURPOSE: To evaluate and compare anticancer therapeutic effect of palmitoyl ascorbate liposomes (PAL) and free ascorbic acid (AA). METHODS: Liposomes incorporating palmitoyl ascorbate (PA) were prepared and evaluated for PA content by HPLC. To elucidate mechanism of action of cell death in vitro, effect of various H(2)O(2) scavengers and metal chelators on PA-mediated cytotoxicity was studied. Effect of various combinations of PAL and free AA on in vitro cytotoxicity was evaluated on 4T1 cells. In vivo, PAL formulation was modified with polyethylene glycol; effect of PEGylation on in vitro cytotoxicity was evaluated. Biodistribution of PEG-PAL formulation was investigated in female Balb/c mice bearing murine mammary carcinoma (4T1 cells). In vivo anticancer activity of PEG-PAL (PEG-PAL equivalent to 20 mg/kg of PA injected intravenously on alternate days) was compared with free AA therapy in same model. RESULTS: PEG-PAL treatment was significantly more effective than free AA treatment in slowing tumor growth. CONCLUSIONS: Nanoparticle formulations incorporating PA can kill cancer cells in vitro. The mechanism of PA cytotoxicity is based on production of extracellular reactive oxygen species and involves intracellular transition metals.
Asunto(s)
Antioxidantes/administración & dosificación , Antioxidantes/uso terapéutico , Ácido Ascórbico/análogos & derivados , Ácido Ascórbico/administración & dosificación , Ácido Ascórbico/uso terapéutico , Neoplasias Mamarias Animales/tratamiento farmacológico , Animales , Mama/efectos de los fármacos , Mama/patología , Línea Celular Tumoral , Femenino , Liposomas , Lipoilación , Neoplasias Mamarias Animales/patología , Ratones , Ratones Endogámicos BALB CRESUMEN
PURPOSE: To evaluate the potential of palmitoyl ascorbate (PA)-loaded micelles for ascorbate-mediated cancer cell targeting and cytotoxicity. METHODS: PA was incorporated in polyethylene glycol-phosphatidyl ethanolamine micelles at varying concentrations. The formulations were evaluated for PA content by RP-HPLC. A stable formulation was selected based on size and zeta potential measurements. A co-culture of cancer cells and GFP-expressing non-cancer cells was used to determine the specificity of PA micelle binding. In vitro cytotoxicity of the micellar formulations towards various cancer cell lines was investigated using a cell viability assay. To elucidate the mechanism of action of cell death in vitro, the effect of various H(2)O(2) scavengers and metal chelators on PA-mediated cytotoxicity was studied. The in vivo anti-cancer activity of PA micelles was studied in female Balb/c mice bearing a murine mammary carcinoma (4T1 cells). RESULTS: PA micelles associated preferentially with various cancer cells compared to non-cancer cells in co-culture. PA micelles exhibited anti-cancer activity in cancer cell lines both in vitro and in vivo. The mechanism of cell death was due primarily to generation of reactive oxygen species (ROS). CONCLUSIONS: The anti-cancer activity of PA micelles associated with its enhanced cancer cell binding and subsequent generation of ROS.
Asunto(s)
Antineoplásicos/química , Ácido Ascórbico , Micelas , Fosfatidiletanolaminas/química , Polietilenglicoles/química , Rodaminas/química , Animales , Antineoplásicos/farmacología , Ácido Ascórbico/análogos & derivados , Ácido Ascórbico/metabolismo , Ácido Ascórbico/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Quelantes/metabolismo , Técnicas de Cocultivo , Portadores de Fármacos/química , Femenino , Depuradores de Radicales Libres/metabolismo , Peróxido de Hidrógeno/química , Concentración 50 Inhibidora , Ratones , Ratones Endogámicos BALB C , Modelos Animales , Células 3T3 NIH , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Especies Reactivas de OxígenoRESUMEN
siRNA is a powerful tool to control cellular processes at the post-transcriptional level. However, its therapeutic potential is limited because of low stability in biological fluids and the lack of simple and efficient delivery systems. Chemical modification of siRNA could be used to increase its intracellular delivery, but may affect its specific activity. To overcome these obstacles, we suggest a simple and effective system capable of stabilization, delivery, and subsequent release of free active siRNA within cells. With this in mind, we reversibly modified the double-stranded GFP-siRNA with a phosphothioethanol (PE) portion via the reducible disulfide bond and incorporated the resulting siRNA-S-S-PE conjugate in nanosized PEG-PE micelles. In the mixed siRNA-S-S-PE/PEG-PE micelles obtained, siRNA was well-protected against degradation by nucleases for at least 24 h, and was released easily from these nanoparticles in free form in the presence of glutathione (GSH) at a concentration mimicking the intracellular levels. In GFP-C166 endothelial cells, mixed GFP-siRNA-S-S-PE/PEG-PE micelles down-regulate the GFP production 50-fold more effectively than free siRNA. In addition, siRNA-containing micelles showed none of the cytotoxic side effects typical for siRNA delivery systems that are based on electrostatic association of siRNA with cationic carriers. Thus, a reversible siRNA-phospholipid conjugate formulated into mixed micelles with PEG-PE can be an effective, nontoxic system for stabilization and delivery of siRNA.
Asunto(s)
Sistemas de Liberación de Medicamentos , Nanopartículas/química , Fosfolípidos/química , Polietilenglicoles/química , ARN Interferente Pequeño/química , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Estabilidad de Medicamentos , Células Endoteliales/química , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Proteínas Fluorescentes Verdes/antagonistas & inhibidores , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/química , Humanos , Micelas , Estructura Molecular , Tamaño de la Partícula , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/farmacología , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/química , Propiedades de SuperficieRESUMEN
Ascorbate has multiple biological roles and chemical interactions, some of which differ between normal and cancerous tissues. Biological effects of ascorbate depend on concentration, route of exposure, and duration of exposure. High-dose ascorbate acts as a pro-oxidant in tissue fluids and delivers peroxide to tissues and fluids, which is then detoxified by erythrocytes and plasma catalase in normally perfused areas. We have previously shown that nanoparticles incorporating palmitoyl ascorbate (PA) targeted and killed cancer cells in vitro. Here, our studies provide additional indications of the importance of extracellular reactive oxygen species (ROS) in the anti-cancer-toxicity by PA-liposomes. Cell death in vitro can be blocked by catalase, superoxide dismutase, and the thiol reductant TCEP. Intracellullar iron may also play a role. Iron chelation by desferrioxamine inhibited cell death but EDTA did not. Further, the fluorescent marker of ROS production in cells indicated that the PA-liposomes caused an increase in ROS. Fluorescent microscopy of tumor sections taken at 3h after injection of rhodamine-labeled liposomes demonstrated an increased accumulation of PA-liposomes compared to plain liposomes. However, the overall biodistribution of (111)In-labeled PA-liposomes was similar to plain liposomes. PA-liposomes provided substantial anti-tumor activity in vivo and enhanced the anti-cancer activity of liposomally encapsulated paclitaxel. Thus, nanoparticles incorporating PA provide a platform for enhancement of the anti-tumor activity of ascorbate.