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Water-in-fluorocarbon reverse emulsions and microemulsions stabilized by semi-fluorinated amphiphiles derived from the dimorpholinophosphate polar head group, C(n)F(2n+1)(CH(2))(m)OP(O)[N(CH(2)CH(2))(2)O](2) (FnHmDMP), are being investigated as new delivery systems for drugs or genetic materials into the lung. Since information related to the toxicity of fluorinated surfactants is still very limited, we evaluated herein the cytotoxicity of a series of FnHmDMP (n=4, 6, 8 and 10 and m=2, 5, and 11). Both solutions of FnHmDMP in fluorocarbons, and reverse water-in-fluorocarbon emulsions stabilized by FnHmDMP were assessed in order to determine the relation between surfactant structure and cell toxicity, and select the most innocuous emulsifier. A first short-term evaluation on mouse fibroblasts using a viability/cytotoxicity assay indicated that amphiphiles (in solution) with a chain length longer than C12 exhibit less toxicity than amphiphiles with shorter chain. Moreover cytotoxicity decreased also with length of the fluorinated segment. The protective effect of the fluorinated chain was strongly supported by the fact that the hydrogenated analog, C(15)H(31)OP(O)[N(CH(2)CH(2))(2)O](2) (H15DMP), was highly toxic. Qualitative evaluation on human lung epithelial cells (HLEC) using a colorimetric method (Mayer's hematoxylin) confirmed that amphiphiles (in solution) with longer chain were the least cytotoxic. The protective effect of the fluorinated chain appeared, however, to be significant only at low amphiphile concentrations (0.1% w/v). In contrast, at higher concentrations (1% and 5% w/v), the total chain length was the determining factor. Quantitative evaluation of the least cytotoxic amphiphiles using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method then showed that F10H11DMP (in solution) was harmless until its solubility limit (1% w/v); cell growth was even enhanced due to improved oxygenation provided by the fluorocarbon phase. F8H11DMP exhibited some cytotoxicity at both 1% and 5% w/v, but the toxicity appeared to level off with concentration. Reverse water-in-perfluorooctyl bromide (PFOB) emulsions stabilized by either F10H11DMP or F8H11DMP were found to be non-cytotoxic. In conclusion, the present evaluation indicates that the cytotoxicity of FnHmDMP depends on both total and fluorinated amphiphile chain length, and leads us to select F8H11DMP and F10H11DMP as the less cytotoxic amphiphiles among a series of FnHmDMP compounds. Furthermore, water-in-fluorocarbon emulsions stabilized with F8H11DMP and F10H11DMP appeared to be non-cytotoxic towards HLEC in culture.

Samples of two Phlebotomus sergenti natural populations from San Juan (Tenerife), representing the western edge of the distribution area of this species, and Axos (Crete) were collected. The morphological comparison showed marked differences in the lengths of parts of the male genitalia, female pharynx, and spermathecae. The isoenzyme study revealed characteristic monomorphic phenotypes for glucose phosphate isomerase, hexokinase, and phosphoglucomutase in the Canarian specimens as compared with the Cretan population. These results confirm the heterogeneity of P. sergenti and indicate the utility of a systematic double approach for a revision of this taxon.

Polyethylenimine (PEI) is one of the most efficient nonviral vectors for gene therapy. The aim of this study was to investigate the role of endocytosis in the transfection of synchronized L929 fibroblasts by PEI/DNA complexes. This was performed by confocal microscopy and flow cytometry, using the endocytosis marker FM4-64 and PEI/DNA complexes labeled either with the DNA intercalator YOYO-1, or with fluorescein covalently linked to PEI. Endocytosis appeared as the major if not the sole mode of entry of the PEI/DNA complexes into the L929 cells. The complexes followed a typical fluid phase endocytosis pathway and were efficiently taken up in less than 10 min in endosomes that did not exceed 200 nm in diameter. Later, the localization of the complexes became perinuclear and fusion between late endosomes was shown to occur. Comparison with the intracellular trafficking of the same complexes in EA.hy 926 cells (W.T. Godbey, K. Wu, A.G. Mikos, Proc. Natl. Acad. Sci. USA 96 (1999)) revealed that endocytosis of PEI/DNA complexes is strongly cell-dependent. In L929 cells, escape of the complexes from the endosomes is a major barrier for transfection. This limited the number of transfected cells to a few percent, even though an internalization of PEI/DNA complexes was observed in most cells. In addition, the entry of the complexes into the nucleus apparently required a mitosis and did not involve the lipids of the endosome membrane. This entry seems to be a short-lived event that involves only a few complexes.

International audience; The aim of this work was to examine the possible influence of the integrity of the microtubule network on the plasma membrane fluidity of L929 mouse fibroblasts. The L929 cell line was selected for the ease of culture and the stability of its characteristics. The cells were treated with colchicine, nocodazole and vinblastine, three microtubule-depolymerizing drugs, at various concentrations and for various times. Membrane fluidity was assessed from fluorescence depolarization measurements with the plasma membrane probe TMA-DPH. Each of the drugs induced a significant, dose-dependent decrease in fluorescence anisotropy. The effect levelled off (5-7% decrease) after approximately 90 min of treatment, and could be unambiguously interpreted as resulting from an increase in membrane fluidity. The cumulative action of the drugs did not significantly increase the effect. The effects of colchicine and nocodazole could be reversed by incubation in drug-free medium, but not that of vinblastine. The results are discussed in correlation with the kinetics of the three drugs interaction with tubulin or microtubules. It is concluded that the microtubule integrity contributed to the high plasma membrane lipidic order, but less than other factors, like the lipid composition and the cholesterol content.

International audience