To further characterize the liposome uptake, kinetic measurements were made using flow cytometry in which 27 mol % DSPE-ATA liposomes were prepared in 150 mM NaCl, phosphate buffer, pH 7.4 along with dipalmitoylphosphatidylglycerol/DPPC/CHOL (50/1733) liposomes in the same buffer. The liposomes were loaded with 6-carboxyfluorescein diacetate (6-CFDA) by lowering the pH to 5.0 and adding the 6-CFDA dissolved in dimethylsulfoxide (DMSO). This fluorophore is non-fluorescent in the diacetate form. Deacetylation resulting from either base hydrolysis or enzymatic hydrolysis yields the fluorescent compound 6-carboxyfluorescein. The fluorophore was found to be stably entrapped in buffer for at least 7 days and was stably entrapped in the presence of serum for 3 hours at 37° C. Both formulations of 6-CFDA labeled liposomes were incubated with 72 hour phytahemaglutanin stimulated peripheral blood mononuclear cells (PBMCs) and the kinetics of liposome uptake were followed by flow cytometry for 48 hours. The results showed that both monocytes and lymphocytes took up the 27 mol % NEST-PE liposomes better than the 50 mol % DPPG liposomes. The results also showed that the kinetics of uptake for the lymphocytes was slower and saturated later than the monocyte liposome uptake.
