Purpose. Knowledge about the uptake mechanism and subsequent intracellular routing of non-viral gene delivery systems is important for the development of more efficient carriers. In this study we compared two established cationic polymers pDMAEMA and PEI with regard to their transfection efficiency and mechanism of cellular uptake. Materials and Methods. The effects of several inhibitors of particular cellular uptake routes on the uptake of polyplexes and subsequent gene expression in COS-7 cells were investigated using FACS and transfection. Moreover, cellular localization of fluorescently labeled polyplexes was assessed by spectral fluorescence microscopy.Results. Both pDMAEMA-and PEI-complexed DNA showed colocalization with fluorescently-labeled transferrin and cholera toxin after internalization by COS-7 cells, which indicates uptake via the clathrinand caveolae-dependent pathways. Blocking either routes of uptake with specific inhibitors only resulted in a marginal decrease in polyplex uptake, which may suggest that uptake routes of polyplexes are interchangeable. Despite the marginal effect of inhibitors on polyplex internalization, blocking the caveolae-mediated uptake route resulted in an almost complete loss of polyplex-mediated gene expression, whereas gene expression was not negatively affected by blocking the clathrin-dependent route of uptake. Conclusions. These results show the importance of caveolae-mediated uptake for successful gene expression and have implications for the rational design of non-viral gene delivery systems.

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Background:To improve the efficiency of liposomal drug targeting systems, it is necessary to understand the mechanism of liposome uptake by the cell and to follow the intracellular fate of internalized liposomes and their contents. Methods: We applied multiple-color fluorescence imaging spectroscopy, using a combination of five fluorescent dyes with a significant spectral overlap. pH-sensitive liposomes were labeled with the hydrophilic dye fluorescein isothiocyanate-dextran (FITC-dextran) or the lipophilic membrane marker rhodamine-B-phosphoethanolamine (Rh-PE) and incubated with COS-7 cells. Further, the cells were stained with specific markers: the cell membrane was fluorescently labeled with Vybrant DiO, lysosomes were stained with LysoTracker Red, and 4Ј,6 diamidino-2-phenylindole dihydrochloride was used for counterstaining the nucleus. Results: All five dyes were used simultaneously and were spectrally distinguished by the system. FITC-dextran-la-

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