Françoise Lavialle

Nanovesicles released by Dictyostelium cells: A potential carrier for drug delivery

Nanovesicles released by Dictyostelium discoideum cells grown in the presence of the DNA-specific dye Hoechst 33342 have been previously shown to mediate the transfer of the dye into the nuclei of Hoechst-resistant cells. The present investigation extends this work by conducting experiments in the presence of hypericin, a fluorescent therapeutic photosensitizer assayed for antitumoral photodynamic therapy. Nanovesicles released by Dictyostelium cells exhibit an averaged diameter between 50 and 150 nm, as measured by transmission cryoelectron microscopy. A proteomic analysis reveals a predominance of actin and actin-related proteins. The detection of a lysosomal membrane protein (LIMP II) indicates that these vesicles are likely generated in the late endosomal compartment. The use of the hypericin-containing nanovesicles as nanodevices for in vitro drug delivery was investigated by fluorescence microscopy. The observed signal was almost exclusively located in the perinuclear area of two human cell lines, skin fibroblasts (HS68) and cervix carcinoma (HeLa) cells. Studies by confocal microscopy with specific markers of cell organelles, provided evidence that hypericin was accumulated in the Golgi apparatus. All these data shed a new light on in vitro drug delivery by using cell-released vesicles as carriers.

Dictyostelium Extracellular Vesicles Containing Hoechst 33342 Transfer the Dye into the Nuclei of Living Cells: A Fluorescence Study

Cells of the eukaryotic unicellular microorganism Dictyostelium discoideum are constitutively resistant to vital staining of their nuclei by the DNA-specific dye Hoechst 33342. By studying the mechanisms of this resistance, we evidenced that these cells expel vesicles containing the dye for detoxification (Tatischeff et al., Cell Mol Life Sci, 54: 476–87, 1998). The question to be addressed in the present work is the potential use of these extracellular vesicles as a biological drug delivery tool, using Hoechst 33342 as a model of a DNA-targeting drug. After cell growth with or without the dye, vesicles were prepared from the cell-free growth medium by differential centrifugation, giving rise to two types of vesicles. Negative staining electron microscopy showed their large heterogeneity in size. Using fluorescence techniques, data were obtained on the dye loading and its environment inside the vesicles. By UV video-microscopy, it was demonstrated that the dye-containing vesicles were able to deliver it into the nuclei of naive Dictyostelium cells, thus overcoming their constitutive resistance to the free dye. A vesicle-mediated dye-transfer into the nuclei of living human leukaemia multidrug resistant K562r cells was also observed.