Evaluation of cytotoxicity of new semi-fluorinated amphiphiles derived from dimorpholinophosphate.

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.

Role of endocytosis in the transfection of L929 fibroblasts by polyethylenimine/DNA complexes.

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.

The influence of microtubule integrity on plasma membrane fluidity in L929 cells.

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.

Cell surface membrane homeostasis and intracellular membrane traffic balance in mouse L929 cells.

We have developed a simple method for synchronizing L929 mouse fibroblasts. Cultured as monolayers, these cells stop growing at confluency and arrest at the end of the G1 phase. Upon seeding at low density, they enter the S phase simultaneously. Using these cells we then looked at the evolution of the surface membrane area during the cell cycle using the fluorescence membrane probe TMA-DPH. In contact with cells, this probe partitions between the membrane (probe fluorescent) and the external medium (non-fluorescent), delivering a signal proportional to the membrane area. This area was constant until just before mitosis, when it increased at once. With the same probe as an endocytic marker, we examined how this membrane homeostasis could be consistent with intracellular membrane trafficking. The study was limited to one selected period of the cell cycle (6-9 hours). We observed that 14% of the membrane endocytosed was not recycled, but was replaced at the cell surface by newly formed membrane from biosynthetic pathways. Brefeldin A modified the membrane traffic, but not the overall membrane homeostasis. The results are discussed in the framework of a maturation model.

[Tests for evaluating the effectiveness of pediculicides: importance and limitations]. / Tests d'évaluation de l'efficacité des pédiculicides : intérêt et limites.

The obligation to prove pediculicides efficacy is relatively recent. Two tests are required by the authorities to obtain registration. In vitro test (with Pediculus humanus humanus) is the first step to evaluate the efficacy of new molecules. It must be followed by bio-clinical tests (with infested children by Pediculus humanus capitis). For those tests the authors advise to respect the "three units rule": unit of time (no more than 2 days for the test), unit of place (same environment) and unit of action (only one team to apply and evaluate products). This type of tests is possible only in countries with high infestation (at least 60% of prevalence). A third test is consequently advised, the ex vivo test using Pediculus humanus capitis obtained from infested children in France. This test can be useful also to re-evaluate current products in the light of increased resistance or decreased sensitivity of the lice. Finally some other tests can be advised to prove some other activities like remanence or repellent properties or to estimated lice resistance.

Phlebotomus sergenti parrot, 1917: morphological and isoenzymatic comparisons of two natural populations from Tenerife (Canary Islands, Spain) and Crete (Greece).

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.