Dye-encapsulating liposomes as fluorescence-based oxygen nanosensors

Nanometer-sized liposomes containing the oxygen-sensitive indicator tris(1,10-phenanthroline)ruthenium chloride (Ru(phen)(3)) in their internal compartment have been prepared and tested for their oxygen-sensing capabilities in aqueous solution. A standard injection technique, where a lipid mixture consisting of dimyristoylphospatidylcholine, cholesterol, and dihexadecyl phosphate (molar ratio 5:4:1) all dissolved in dry 2-propanol injected into an aqueous solution of 10 mM Ru(phen)(3) under vortexing, is used to prepare the liposomes. A high uniformity of the liposomes is realized by extruding them back and forth through a 100-nm pore size polycarbonate membrane. TEM images of the liposomes, stained with uranyl acetate, show that the liposomes are unilamellar, round in shape, maintain high structural integrity, and average 70 mn in diameter. Dynamic light-scattering measurements support-this observation. Under our experimental conditions, the entrapment efficiency of Ru(phen)(3), defined as the ratio between the concentration of dye molecules encapsulated in the liposomes and the dye concentration in the solution used for liposome formation, is similar to 1%, The liposomes show high stability with respect to dye leaking at room temperature for 8 days and high photostability when exposed to the excitation light. Individual liposomes are used to monitor the enzymatic oxidation of glucose by glucose oxidase in their vicinity. The newly prepared oxygen-sensitive liposomes can be applied for noninvasive oxygen analysis in tissues and single biological cells.