Liposome-based optochemical nanosensors

This paper describes the optochemical pH and oxygen sensing properties of d ye-encapsulating and fluorescently labeled nano-sized unilamellar liposomes . To prepare the oxygen sensitive liposomes a lipid mixture consisting of d imyristoylphospatidylcholine, cholesterol, and dihexadecyl phosphate (molar ratio 5:4:1) all dissolved in dry isopropyl alcohol is injected into a sen sing dye solution. The mixture is then sonicated with a liposome maker to f orm dye-encapsulating liposomes. A lipid mixture consisting of dimyristoylp hospatidylcholine, N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-gl ycero-3-phosphoethanolamine triethylammonium salt (fluorescein DHPE), chole sterol, and dihexadecyl phosphate (molar ratio 20:1:16:4) is used to prepar e the pH sensitive liposomes by the same sonication technique. Fluorescein labeled DHPE phospholipids are combined with DMPC phospholipids in a 1:20 r atio to incorporate the sensing dye directly into the bilayer membrane, vir tually eliminating any instability due to dye leakage. Oxygen sensing lipos omes are created by encapsulating the oxygen sensitive ruthenium tris(1,10) -phenanthroline complex [Ru(phen)(3)]. The dye is believed to exist both in free solution within the liposome, and as an adherent on the inner membran e of the liposome. High uniformity of the liposomes is realized by extrudin g them back and forth through a 100 nm pore-size polycarbonate membrane. TE M images of the liposomes, stained with uranyl acetate, show that the lipos omes are unilamellar, spherical in shape, maintain high structural integrit y, and average 70 nm in diameter. The liposomes show high stability with re spect to dye leaking at room temperature for 8 days, and high photostabilit y when exposed to the excitation light. Individual liposomes are used to mo nitor the pH and oxygen level in their vicinity during the enzymatic oxidat ion of glucose by the enzyme glucose oxidase. The newly prepared environmen tally sensitive liposomes can be applied for non-invasive pH and oxygen det ermination in tissues and single biological cells.