Liposomal preparation for the in vivo monitoring of osmolality

We wished to develop a tool to monitor changes in our in vivo microvascular preparations. Our criteria were to have the probe be biocompatible, emit a reproducible signal from a microvascular compartment (vessel or lymphatic) as a function of osmotic pressure, and posses the means to monitor the probe concentration. We chose to use liposomes containing a fluorescent dye excited at visible wavelengths at a concentration that was self‐quenched, resulting in increasing fluorescence intensity (FI) with decreasing osmolality. A lipid film was made by combining 1,2‐Diestearoyl‐ sn ‐Glycero‐3‐Phosphotidyl choline (DSPC), cholesterol, and 18:0 PEG(2000)PE (DSPE‐PEG) (Avanti Polar Lipids) at a molar ratio of 1:0.3:0.08 (Jayaraman, S., J. Gen. Physiol., 2001) in 2 ml ethanol. Following ethanol evaporation the lipid film was rehydrated with 2 ml buffer (pH 7.4, 300 mOsm) containing 4.7 mM KCL, 2.0 mM CaCl 2 , 1.2 mM MgSO 4 , 1.2 mM NaH 2 PO 4 , 3.0 mM NaHCO 3 , 111 mM NaCl, and 30 mM Calcein. Liposomes of uniform size were created with an Avanti mini‐extruder fitted with 0.4 μm pore polycarbonate membrane filters. Unencapsulated dye was removed by size exclusion chromatography over Sephacryl S‐200 HR (GE Healthcare). After brief equilibration over a range of osmolalities, (264–326 mOsm) the liposomes were excited at 480 nm and emission was monitored at 530 nm. The FI was inversely related to osmolality (r 2 =0.99). Absorbance read at 480 nm did not change over the same range of osmolalities (slope not different from 0, p>0.5). The stability of the absorbance value allows it to be used as a reference for the calibration of FI to osmolality. Finally, as the ratio of absorbance to FI was related directly to osmolality (r 2 =0.99) these liposomes can be used to monitor quantitatively osmotic changes in vivo. Supported by NIH HLO78816, NASA NN04GA50G, AHA 0615515Z (RS), and NIHCO6RRO17353