Purpose: We have previously described a new multi-target drug named nitrooxy-doxorubicin (Nit-DOX) that exhibits a novel and unexpected mechanism of action and high anticancer activity. In this work we show the results obtained with the preparation of liposomal formulation of the compound in order to improve its delivery and its target-ability. Methods: Liposomal Nit-DOX was prepared by thin lipid film hydration and extrusion method and characterized in terms of size and surface charge. Drug release into phosphate buffer pH 7.4 and serum was evaluated. In vitro cytotoxicity, intracellular doxorubicin accumulation and nitric oxide release were evaluated in human colon cancer chemosensitive HT29 cells, in their resistant counterpart HT29-dx (which express high levels of drug efflux transporters, such as P-glycoprotein and multidrug resistance related protein 1), and in rat cardiomyocytes H9c2. Results: Two liposomal Nit-DOX formulation containing different amounts of cholesterol (15 and 30%) were prepared. They showed similar size and Z potential values, while the amount of Nit-DOX encapsulated increased with the decreasing of the rate of cholesterol. The formulation were stable for more than 28 days in buffer at 4°C, retained 90% of their initial content and no drug precipitation or liposomes aggregation were observed. The drug leakage was higher in buffer and serum at 37°C with the liposomes containing 15% of cholesterol. The biological data showed that the in vitro efficacy of Nit-DOX was further increased when the drug was encapsulated in liposomes. The liposomal formulation showed a higher cytotoxicity, a prolonged intracellular retention of doxorubicin and a more sustained release of nitric oxide than free Nit-DOX or parental DOXO in both HT29 and HT29-dx cells. Moreover liposomal Nit-DOX exhibited a significantly lower cytotoxicity than free DOXO in cardiomyocytes. Conclusion: We were able to obtain liposomal formulation that permitted to increase the stability and the cytotoxicity of Nit-DOX. Studies are in progress to further investigate the molecular mechanisms by which this system overcomes drug resistance.
PREPARATION AND CHARACTERIZATION OF LIPOSOMES CONTAINING A NEW ANTHRACYCLINE DERIVATIVE
PEDRINI, ISABELLA;GAZZANO, Elena;ARPICCO, Silvia Maria;RIGANTI, Chiara;ROLANDO, Barbara;CHEGAEV, Konstantin;FRUTTERO, Roberta;GASCO, Alberto
2013-01-01
Abstract
Purpose: We have previously described a new multi-target drug named nitrooxy-doxorubicin (Nit-DOX) that exhibits a novel and unexpected mechanism of action and high anticancer activity. In this work we show the results obtained with the preparation of liposomal formulation of the compound in order to improve its delivery and its target-ability. Methods: Liposomal Nit-DOX was prepared by thin lipid film hydration and extrusion method and characterized in terms of size and surface charge. Drug release into phosphate buffer pH 7.4 and serum was evaluated. In vitro cytotoxicity, intracellular doxorubicin accumulation and nitric oxide release were evaluated in human colon cancer chemosensitive HT29 cells, in their resistant counterpart HT29-dx (which express high levels of drug efflux transporters, such as P-glycoprotein and multidrug resistance related protein 1), and in rat cardiomyocytes H9c2. Results: Two liposomal Nit-DOX formulation containing different amounts of cholesterol (15 and 30%) were prepared. They showed similar size and Z potential values, while the amount of Nit-DOX encapsulated increased with the decreasing of the rate of cholesterol. The formulation were stable for more than 28 days in buffer at 4°C, retained 90% of their initial content and no drug precipitation or liposomes aggregation were observed. The drug leakage was higher in buffer and serum at 37°C with the liposomes containing 15% of cholesterol. The biological data showed that the in vitro efficacy of Nit-DOX was further increased when the drug was encapsulated in liposomes. The liposomal formulation showed a higher cytotoxicity, a prolonged intracellular retention of doxorubicin and a more sustained release of nitric oxide than free Nit-DOX or parental DOXO in both HT29 and HT29-dx cells. Moreover liposomal Nit-DOX exhibited a significantly lower cytotoxicity than free DOXO in cardiomyocytes. Conclusion: We were able to obtain liposomal formulation that permitted to increase the stability and the cytotoxicity of Nit-DOX. Studies are in progress to further investigate the molecular mechanisms by which this system overcomes drug resistance.
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