Investigating the uptake and intracellular fate of pH-sensitive liposomes by flow cytometry and spectral bio-imaging.

The intracellular fate of particulate drug delivery systems is determined by the initial mode of internalization and the subsequent intracellular trafficking. Besides clathrin-mediated endocytosis, different cellular uptake mechanisms for internalization are discussed, including caveolae-mediated endocytosis, phagocytosis, macropinocytosis and the badly characterized non-clathrin-non-caveolae-mediated endocytosis. In addition, fusion processes could also be included. For a targeted drug delivery, the mechanism of caveolae uptake seems to be most promising since it does not transport internalized material to endosomes and lysosomes where internalized material might be degraded. The knowledge of the mode of internalization is therefore the first step for optimized drug targeting. The uptake and the intracellular behavior of pH-sensitive liposomes are investigated in two different cell types. For investigation, two different approaches were taken: a) a combination of different inhibitors was used to selectively block different pathways. These effects were evaluated by flow cytometry. b) Fluorescently labeled markers, specific for the different endocytic routes, were incubated with fluorescently labeled liposomes with a subsequent check for co-localization (spectral bio-imaging). COS-7 and HUVEC internalize pH-sensitive liposomes by means of different mechanisms. HUVEC seem to internalize via clathrin-dependent endocytosis, caveolae-dependent endocytosis and macropinocytosis whereas COS-7 cells behave differently: here macropinocytosis is not involved in the uptake. We show that the combination of flow cytometry and spectral bio-imaging offers the possibility to understand the initial mode of internalization and to follow the intracellular fate of liposomes, both of which are a prerequisite for optimizing drug targeting systems.