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

https://doi.org/10.1016/j.ijpharm.2009.06.039Get rights and content

Abstract

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.

Introduction

Efficient internalization of therapeutic agents within cells is required to successfully achieve both conventional drug mediated-therapy and gene-therapy. The rational for our study is the search for overcoming the general cell resistance towards cell penetration of therapeutic drugs, as previously demonstrated with a DNA-specific drug (Tatischeff et al., 2008).

Besides viral-designed vectors, an assortment of non-viral tools, including liposomes, polymers and nanoparticles, have been proposed to vectorize therapeutic agents both in vitro and in vivo (Derycke and De Witte, 2002, Torchilin, 2005, Delcayre and Le Pecq, 2006, Weissig et al., 2006). We have previously shown that cells of Dictyostelium discoideum, a non-pathogenic eukaryotic amoeba, constitutively release nanovesicles (Tatischeff et al., 1998, Tatischeff et al., 2008). When cell growth is initiated in the presence of exogenous molecules, such as a DNA-specific dye, Hoechst 33342 (HO342), Dictyostelium cells produce dye-loaded nanovesicles as a detoxification mechanism (Tatischeff et al., 1998). These nanovesicles can transfer their cargo to the nuclei of naive Dictyostelium and human leukeamia K562r resistant cells, which are both resistant to the vital labeling of their nuclei by the dye (Tatischeff et al., 2008). Consequently, we proposed that these nanovesicles of biological origin could also be loaded with therapeutic molecules and then used as a nanodevice for cellular drug internalization.2

In the fight against cancer, photodynamic therapy is a non-invasive technique, which involves the systemic administration of a photosensitizer and the local irradiation of the tumor tissue with visible light in order to generate highly cytotoxic reactive oxygen species. Among available photosensitizers, the polycyclic anthraquinone hypericin, a natural pigment present in Hypericum perforatum is endowed with promising properties for photodiagnosis and photodynamic therapy of cancer (Agostinis et al., 2002, Head et al., 2006, Ritz et al., 2007, Buytaert et al., 2008, Seitz et al., 2007, Seitz et al., 2008). However, hypericin is a lipophilic non-hydrosoluble drug, making its intraveinous injection problematic (Van De Putte et al., 2006). To overcome this difficulty and to optimize therapeutic effect of the agent, different delivery systems have been developed in vitro (see Saw et al., 2006 for review), such as polyvinylpyrrolidone binding, LDL-assisted endocytosis or embedding in sterically stabilised PEG-liposomes (Derycke and De Witte, 2002). When considering in vivo applications, the fast elimination of liposomes from the blood has been overcome by different sterical stabilizations (Torchilin, 2005). However, some drawbacks of the carrier-systems remain, such as the loss of substantial amount of hypericin from the liposomes (Derycke and De Witte, 2002), followed by potential harmful aggregation in physiological media.

In our study, hypericin was chosen as an antitumoral compound of interest to promote the nanovesicles released by Dictyostelium cells as drug carriers. After studying the loading capacity of these nanovesicles with the hydrophobic photosensitizer, an in vitro vesicle-mediated hypericin transfer assay was successfully conducted on two human cell lines, HS68 skin fibroblasts and carcinomic HeLa cells. The results point out the vesicles from Dictyostelium cells as a new interesting bio-engineered carrier device for drug delivery.

Section snippets

Materials and methods

If not otherwise specified, chemical reagents were from Sigma (L’Isle d’Abeau Chesnes, France) of the highest available purity.

Dictyostelium cells produce nanovesicles loaded with hypericin

Dictyostelium cells are capable to internalize exogenous molecules and to get rid of them as cargo molecules packaged into nanovesicles released in the external medium. With the aim to use these nanovesicles as a vector for therapeutic drugs, we carried out assays with hypericin, a promising photosensitizer for phototherapy. If light-dependent toxicity of hypericin is well known (Ritz et al., 2007, Wielgus et al., 2007), the molecule has also been suggested to have noticeable cytotoxic effects

Discussion

The present study was initiated to investigate the potential use of Dictyostelium cell-released nanovesicles as in vitro drug carriers for cancer therapy. It extends our previous investigation using HO342-loaded nanovesicles, that allowed to label the nuclei of naive Dictyostelium cells and human leukaemic K562r cells, even though these two cell types are constitutively resistant to the vital staining of their nuclei (Tatischeff et al., 2008). The present results indicate that Dictyostelium

Conclusion

Our work indicates that D. discoideum is a bio-engineering designer able to formulate vesicular drug carriers. To our knowledge, this is the first study describing cell-engineered vesicles able to load and vectorize a therapeutic molecule within human cells, as shown with the important photosensitizer, hypericin.

With regard to the possibility of using Dictyostelium nanovesicles in therapy, many problems remain to be solved. A study to elucidate the membrane events involved in cell entry of the

Acknowledgements

We would like to thank Gilles Lemaitre (istem, Evry, France) for help with the SDS-PAGE; Jean-Michel Guignet (CNRS UMR 7590, Paris, France) for measurements of the nanovesicle diameters and C. Lavialle (CNRS UMR 8122, Villejuif, France) for critical reading of the manuscript.

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