Dissociation of fluorescently labeled lipids from liposomes in biological environments challenges the interpretation of uptake studies

Within nanomedicine, liposomes are investigated for their ability to deliver drug cargoes specifically into subcellular compartments of target cells. Such studies are often based on flow cytometry or microscopy, where researchers rely on fluorescently labeled lipids (FLLs) incorporated into the liposomal membrane to determine the localization of the liposomes within cells. These studies assume that the FLLs stay embedded in the liposomal membrane throughout the duration of the experiment. Here, we used size exclusion chromatography (SEC) to investigate the validity of this assumption by quantitatively determining the propensity of various widely used FLLs to dissociate from liposomes during incubation in human plasma. For certain commonly used off-the-shelf FLLs, up to 75% of the dye dissociated from the liposomes, while others dissociated less than 10%. To investigate the implications of this finding, we measured the peripheral blood leukocyte uptake of liposomes formulated with different FLLs using flow cytometry, and observed a significant difference in uptake correlating with the FLL's dissociation tendencies. Consequently, the choice of FLL can dramatically influence the conclusions drawn from liposome uptake and localization studies due to uptake of dissociated FLLs. The varying dissociation propensities for the FLLs were not reflected when incubating in buffer, showing that non-biological environments are unsuitable to mimic liposomal stability in a drug delivery context. Overall, our findings suggest that it is crucial for researchers to evaluate the stability of their FLL-labeled liposomes in biological environments, and the simplicity of the SEC assay put forward here makes it very applicable for the purpose.