RESUMO
Arsonoliposomes (ARSL) are liposomes that incorporate arsonolipids (ARS) in their membranes. They have demonstrated significant toxicity towards cancer cells, while being less toxic towards normal cells. In this study, we sought to investigate the possibility to prepare novel types of arsonoliposomes (ARSL) by incorporating a lipidic derivative of curcumin (TREG) in their membrane, and/or by loading the vesicles with doxorubicin (DOX). The final aim of our studies is to develop novel types of ARSL with improved pharmacokinetics/targeting potential and anticancer activity. TREG was incorporated in ARSL and their integrity during incubation in buffer and serum proteins was studied by monitoring calcein latency. After evaluation of TREG-ARSL stability, the potential to load DOX into ARSL and TREG-ARSL, using the active loading protocol, was studied. Loading was performed at two temperatures (40 °C and 60 °C) and different time periods of co-incubation (of empty vesicles with DOX). Calculation of DOX entrapment efficiency (%) was based on initial and final drug/lipid ratios. The cytotoxic activity of DOX-ARSL was tested towards B16F10 cells (mouse melanoma cells), LLC (Lewis Lung carcinoma cells), and HEK-293 (Human embryonic kidney cells). Results show that TREG-ARSL have slightly larger size but similar surface charge with ARSL and that they are both highly stable during storage at 4 °C for 56 d. Interestingly, the inclusion of TREG in ARSL conferred increased stability to the vesicles towards disruptive effects of serum proteins. The active-loading protocol succeeded to encapsulate high amounts of DOX into ARSL as well as TREG-LIP and TREG-ARSL, while the release profile of DOX from the novel liposome types was similar to that demonstrated by DOX-LIP. The cytotoxicity study results are particularly encouraging, since DOX-ARSL were less toxic towards the (normal) HEK cells compared to the two cancer cell-types. Furthermore, DOX-ARSL demonstrated lower toxicities (at all concentrations tested) for HEK cells, compared to that of the corresponding mixtures of free DOX and empty ARSL, while the opposite was true for the cancer cells (in most cases). The current results justify further in vivo exploitation of DOX-ARSL, as well as TREGARSL as anticancer therapeutic systems.
RESUMO
Sonicated arsonoliposomes were prepared using arsonolipid with palmitic acid acyl chain (C16), mixed with phosphatidylcholine (PC)-based or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-based, and cholesterol (Chol) with C16/DSPC/Chol 8:12:10 molar ratio. PEG-lipid (1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated to polyethylenoglycol 2000) containing vesicles (PEGylated-arsonoliposomes; PC-based and DSPC-based) were also prepared. The cytotoxicity of these arsonoliposomes towards different cancer cells (human promyelocytic leukaemia NB4, Prostatic cancer PC3, human breast adenocarcinoma MDA-MB-468, human T-lymphocyte (MT-4) and also towards human umbilical vein endothelial cells (HUVECs) was evaluated by calculating the arsonoliposome-induced growth inhibition of the cells by the MTT assay. IC-50 values were interpolated from cell number/arsonoliposome concentration curves. The results reveal that all types of arsonoliposomes evaluated significantly inhibit the growth of most of the cancer cells studied (PC3, NB4, MT4) with the exception of the MDA-MB-468 breast cancer cells which were minimally affected by arsonoliposomes; in some cases even less than HUVEC. Nevertheless, for the same cell type the differences between the different types of arsonoliposomes were significant but not proportional to their stability, indicating that the formation of arsonoliposomes with very stable membranes is not a problem for their anticancer activity. Thereby it is concluded that arsonoliposome composition should be adjusted in accordance to their in vivo kinetics and the desired, for each specific application, biodistribution of As and/or encapsulated drug.
Assuntos
Antineoplásicos/farmacologia , Arsenicais/farmacologia , Lipídeos/análise , Lipossomos/farmacologia , Arsenicais/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Lipossomos/químicaRESUMO
Sonicated arsonoliposomes were prepared using arsonolipid with palmitic acid acyl chain (C16), mixed with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC-based), and cholesterol (Chol) with a molar ratio C16/DSPC/Chol 8:12:10. PEG-lipid (1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated to polyethylenoglycol 2000) containing vesicles (Pegylated-arsonoliposomes) were also prepared. DSPC-based and Pegylated-arsonoliposomes, were administered by intraperitoneal injection in balb/c mice (15 mg arsenic/kg) and the distribution of As in the organs was measured by atomic absorption spectroscopy. Results demonstrate that a high portion of the dose administered is rapidly excreted since 1 h post-injection only about 30-40% of the dose was detected cumulatively in animal tissues. After this, the whole body elimination of arsenic was a slow process with a half-life of 27.6 h for Pegylated-arsonoliposomes, and 83 h, for the DSPC-based ones. For both arsonoliposomes, arsenic distribution was greater in intestines, followed by liver, carcass+skin stomach, spleen, kidney, lung and heart. Different arsenic kinetics in blood between the two liposome types were observed. Compared to the results obtained previously with PC-based arsonoliposomes, both the DSPC-based and Pegylated-arsonoliposomes have better bioavailability. This proves that arsonoliposome lipid composition (and consequently their integrity) influences their pharmacokinetic profile. Thus, the proper arsonoliposome composition should be used according to the intended application.
Assuntos
Arsênio/análise , Arsenitos/farmacocinética , Lipídeos/química , Palmitatos/farmacocinética , Animais , Antiparasitários/administração & dosagem , Antiparasitários/química , Antiparasitários/farmacocinética , Arsênio/sangue , Arsenitos/administração & dosagem , Arsenitos/química , Disponibilidade Biológica , Colesterol/química , Feminino , Injeções Intraperitoneais , Lipossomos , Camundongos , Camundongos Endogâmicos BALB C , Palmitatos/administração & dosagem , Palmitatos/química , Tamanho da Partícula , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Eletricidade Estática , Propriedades de Superfície , Distribuição TecidualRESUMO
Sonicated arsonoliposomes were prepared using an arsonolipid with palmitic acid acyl chain (C16), mixed with phosphatidylcholine (PC-based) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC-based), and cholesterol (Chol) with a molar ratio C16 /PC or DSPC/ Chol 8:12:10. PEG-lipid (1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated to polyethylenoglycol 2000) containing vesicles (pegylated-arsonoliposomes) were also prepared. The in vitro and in vivo trypanocidal activity of the various types of arsonoliposomes was evaluated. Although PC-based arsonoliposomes exhibited in vivo activity on an acute trypanosomiasis animal model, no evidence of activity was demonstrated for DSPC-based or pegylated-arsonoliposomes on a chronic model. Despite the fact that DSPC-based and pegylated-arsonoliposomes have better bioavailability compared to PC-based ones, their in vitro activity is lower than that of PC-based arsonoliposomes, indicating the importance of arsonoliposome lipid composition on their trypanocidal activity and suggesting that further arsonoliposome structure design is required to overcome these disadvantages.
Assuntos
Arsenitos/farmacologia , Palmitatos/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Animais , Arsenitos/administração & dosagem , Arsenitos/química , Disponibilidade Biológica , Colesterol , Técnicas In Vitro , Lipossomos , Camundongos , Palmitatos/administração & dosagem , Palmitatos/química , Fosfatidilcolinas , Fosfatidiletanolaminas , Polietilenoglicóis , Tripanossomicidas/administração & dosagem , Tripanossomicidas/química , Trypanosoma brucei brucei/metabolismo , Tripanossomíase/tratamento farmacológicoRESUMO
Increased toxicity of arsonoliposomes towards cancer cells may be attributed to interaction between arsonolipids and cellular thiols which, would result in reduction of As(V) to the more toxic As(Ill). Cancer cells with high thiol contents may thus be more sensitive to arsonoliposomes, providing that the arsonolipid molecules that are incorporated in the liposome membrane can interact with thiol-containing compounds. For examination of this possibility we investigate the effect of incubating various compositions of arsonoliposomes with glutathione, on their integrity. If glutathione does interact with the As(V) of the arsonolipid headgroup, this should result in an alteration of the arsonoliposome membrane stability. We followed arsonoliposome integrity by measuring the release of vesicle-encapsulated calcein from arsonoliposomes with different lipid compositions, during incubation in glutathione. The results of this study show that the effect of glutathione on arsonoliposome integrity is higher (arsonoliposomes are less stable) when the arsonolipid content of their membranes increases. This indicates that arsonolipid molecules interact with glutathione, and in some cases, depending on the rigidity of their membranes; this interaction leads to a (higher or lower) destabilization of arsonoliposomes. The destabilizing effect of glutathione was higher for arsonoliposomes that were previously found to be less stable during incubation in serum proteins or, in other words, have lower membrane rigidity. In the case of pegylated-arsonoliposomes membrane destabilization was minimal and this may be related to the high stability demonstrated previously for these specific arsonoliposomes, or, it may indicate that pegylation results in prevention (total or partial) of arsonolipid-As interaction with thiols (perhaps because of steric repulsion).