Fusion pores with low conductance are cation selective.

Many neurotransmitters are organic ions that carry a net charge, and their release from secretory vesicles is therefore an electrodiffusion process. The selectivity of early exocytotic fusion pores is investigated by combining electrodiffusion theory, measurements of amperometric foot signals from chromaffin cells with anion substitution, and molecular dynamics simulation. The results reveal that very narrow fusion pores are cation selective, but more dilated fusion pores become anion permeable. The transition occurs around a fusion pore conductance of ∼300 pS. The cation selectivity of a narrow fusion pore accelerates the release of positively charged transmitters such as dopamine, noradrenaline, adrenaline, serotonin, and acetylcholine, while glutamate release may require a more dilated fusion pore.

Enhanced killing of human epidermoid carcinoma (KB) cells by treatment with ricin encapsulated into sterically stabilized liposomes in combination with monensin.

Ricin was encapsulated in various charged liposomes having 5 mol% PEG of different chain length on the surface. The cytotoxicity of ricin entrapped in these liposomal formulations was examined in human epidermoid carcinoma (KB) cells with a view to develop an optimum delivery system for ricin in vivo. It was observed that the cytotoxicity of ricin entrapped in various charged liposomes was significantly dependent on the surface charge as well as chain length of PEG. The maximum cytotoxicity of ricin was observed when it was delivered through negatively charged liposomes having 5 mol% PEG-2000 on the surface. Monensin enhances the cytotoxicity of ricin entrapped in various charged liposomes depending on the surface charge. Maximum potentiation of cytotoxicity of ricin was observed when it was delivered through negatively charged liposomes having 5 mol% PEG-2000 on the surface. Studies on the kinetics of inhibition of protein synthesis by ricin revealed that the lag period of inhibition of protein synthesis is significantly lengthened following its delivery through various charged liposomes. Monensin significantly reduced the lag period of action of ricin. It was also observed that the efficacies of monensin on the enhancement of cytotoxicity of ricin entrapped in various charged PEG-liposomes were highly related to their amount of cell association. The current study has demonstrated that by suitable adjustment of charge, density, and chain length of PEG on the surface of liposomes it would be possible to direct liposomal ricin to human tumor cells for their selective elimination in combination with monensin.

Effect of surface charge and density of distearylphosphatidylethanolamine-mPEG-2000 (DSPE-mPEG-2000) on the cytotoxicity of liposome-entrapped ricin: effect of lysosomotropic agents.

Ricin was encapsulated in various liposomes having neutral, negatively and positively charged and different density of DSPE-mPEG-2000 on the surface and cytotoxicity of ricin entrapped in these different charged liposomal formulations was studied in CHO pro(-) cells and compared with free ricin with a view to develop an optimum delivery system for ricin in vivo. It was observed that the cytotoxicity of ricin entrapped in various charged liposomes was significantly dependent on the charge on the surface of liposomes. The maximum cytotoxicity of ricin was observed when it was delivered through negatively charged liposomes. Monensin enhances the cytotoxicity of ricin entrapped in various charged liposomes and the extent of enhancement of the cytotoxicity is significantly dependent on the charge on the surface of liposomes. Maximum potentiation (213.14-fold) of cytotoxicity of ricin was observed when it was delivered through positively charged liposomes followed by negatively charged (83.36-fold) and neutral (71.30-fold) liposomes, respectively. Studies on the kinetics of inhibition of protein synthesis by ricin entrapped in various charged liposomes revealed that lag period of inhibition of protein synthesis is significantly lengthened following delivery through various charged liposomes. However, in the presence of monensin, the lag period was reduced. There is a marginal variation in the cytotoxicity of ricin entrapped in various charged liposomes after incorporation of 5mol% of DSPE-mPEG-2000 on the surface. However, there is a significant variation in the enhancing potency of monensin on the cytotoxicity of ricin entrapped in various charged liposomes in CHO pro(-) cells following incorporation of 5mol% DSPE-mPEG-2000 on the surface. Studies on the effect of variation of density of DSPE-mPEG-2000 on the surface of various charged liposomes on the enhancement of cytotoxicity of entrapped ricin by monensin in CHO pro(-) cells showed that the enhancing potency of monensin on the cytotoxicity of ricin entrapped in various charged liposomes is significantly dependent on the density of DSPE-mPEG-2000 on their surface. It was also observed that the efficacies of monensin on the enhancement of cytotoxicity of ricin entrapped in various charged PEG-liposomes in CHO pro(-) cells was highly related to their amount of cell-association. The present study has clearly shown that by suitable alteration of liposomal lipid composition, charge and density of hydrophilicity it would be possible to direct liposomal ricin to specific cells for their selective elimination in combination with monensin.

Enhancement of the cytotoxicity of liposomal ricin by the carboxylic ionophore monensin and the lysosomotropic amine NH4Cl in Chinese hamster ovary cells.

Ricin was encapsulated in negatively charged liposomes and its effect on the cytotoxicity was compared with native ricin in Chinese hamster ovarian (CHO) cells. The cytotoxicity of ricin, as measured by a marker protein synthesis (incorporation of 3H-leucine), was reduced markedly (300-fold) following encapsulation in liposomes. Lactose, a potent inhibitor of ricin cytotoxicity, had no effect on the binding, internalization, and cytotoxicity of liposomal ricin, indicating that liposomal ricin enter into mammalian cells by an alternative route, bypassing galactose-mediated endocytic pathway. Both monensin (a carboxylic ionophore) and NH4Cl (a lysosomotropic amine) markedly enhances the cytotoxicity of liposomal ricin, indicating endocytotic uptake of liposomal ricin. The degree of potentiation of the cytotoxicity of liposomal ricin by both monensin and NH4Cl was significantly higher (441- and 51-fold) as compared to native ricin (62.5- and 12.5-fold). The extent of exocytosis of free ricin was found to be much higher as compared to liposomal ricin; on the other hand, the extent of degradation of free and liposomal ricin was identical. Consequently, the intracellular level of liposomal ricin was increased to 3.5-fold. This higher level of intracellular liposomal ricin may allow more efficient ricin A-chain release into the cytosol under the influence of NH4Cl and monensin. Monensin-induced potentiation of liposomal ricin was prevented by brefeldin A, indicating that in the presence of monensin, the liposomal ricin was efficiently routed through the Golgi apparatus en route to the cytosol. Thus, liposomal ricin in combination with monensin may have potential application for selective elimination of malignant cells.