An Cao

The osmotic response of large unilamellar vesicles studied by quasielastic light scattering

Large unilamellar vesicles of two phosphatidylcholines, one saturated (DMPC) and the other unsaturated (DOPC), prepared by the reverse-phase evaporation method were studied using the quasielastic light scattering technique. The accurate sizing obtained by this technique showed an osmotic response for the two kinds of vesicles when the salinity of the external medium was diluted. The elastic moduli of lipid vesicles bilayers in the liquid phase were then estimated according to the elasticity theory of spherical shells taking into account salt leakage data known from the literature.

Evaluation and optimization of DNA delivery into gliosarcoma 9L cells by a cholesterol-based cationic liposome

This paper reports results concerning the transfection of gliosarcoma cells 9L using an original cholesterol-based cationic liposome as carrier. This cationic liposome was prepared from triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol) and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE). The used concentration of liposome was not cytotoxic as revealed by the MTT test. TEAPC-Chol/DOPE liposomes allowed the plasmids encoding reporter genes to enter the nucleus as observed both by electron microscopy and functionality tests using fluorescence detection of green fluorescent protein (GFP) and luminometric measurements of luciferase activity. By changing the cationic lipid/DNA molar charge ratio, optimal conditions were determined. Further, improvement of the transfection level has been obtained by either precondensing plasmid DNA with poly-L-lysine or by adding polyethylene glycol (PEG) in the transfection medium. The optimal conditions determined are different depending on whether the transfection is made with cells in culture or with tumors induced by subcutaneous (s.c.) injection of cells in Nude mice. For in vivo assays, a simple method to overcome the interference of haemoglobin with the chemiluminescence intensity of luciferase has been used. These results would be useful for gaining knowledge about the potential for the cationic liposome TEAPC-Chol/DOPE to transfect brain tumors efficiently.

Modifications in the Head Group and in the Spacer of Cholesterol-based Cationic Lipids Promote Transfection in Melanoma B16-F10 Cells and Tumours

A series of four cationic lipids derived from cholesterol was synthesised and their efficiencies to vectorise nucleic acids were compared. The investigation concerns the effects of systematic chemical modifications in the polar head and in the spacer. The cationic lipid molecules used are in the same family of 3β[N-(N′,N′,N′-trimethylaminoethane)-carbamoyl] cholesterol iodide (TMAEC-Chol), presenting a spacer of two or three carbons and a quaternary ammonium polar head ramified with methyl or ethyl groups. These lipids formed stable liposomes sizing from 100 to 200 nm when prepared with the colipid dioleoyl phosphatidylethanolamine (DOPE). The goal of this work was to investigate the effect of the chemical structure of these cationic lipids on lipofection. Their ability to form complexes with DNA, their cytotoxicity and their transfection efficiency in vitro and in vivo were studied. Results were compared with those obtained from the well known cholesterol-based cationic lipid DC-Chol. In a melanoma cell line (B16-F10), results showed that either the polar head or the spacer affected the cytotoxicity. Cationic lipids with three ethyl groups in the head are more toxic than those with three methyl groups while cationic lipids with three carbons in the spacer are less toxic than those with two carbons in the spacer. The best transfection level was obtained in vitro and in vivo with cationic lipids having 3C in the spacer. Data indicated that among these lipids, in vivo gene transfer is advantaged by the methylated polar head while in vitro the best level was obtained with the ethylated one. Finally, it was observed that the chemical structure influences the transfection in the presence of serum while the complex charge and the DOPE ratios in liposomes preferentially affect the interaction with erythrocytes. Argumentations are proposed to explain the discrepancies between in vitro and in vivo transfection results concerning the optimal charge ratio and the chemical nature of the cationic lipid head group.

Lipopeptide-based liposomes for DNA delivery into cells expressing neuropilin-1

In this paper, liposomes containing a lipopeptide bearing a ligand specifically recognized by neuropilin-1 (NRP-1) have been used to target a human breast cancer cell line overexpressing this receptor. The synthesis of this lipopeptide, C16-A7R, formed by the sequence of 7 aminoacids ATWLPPR, linked to a palmitoyl fatty chain by an amide bond was described. After the characterisation of cationic liposomes formulated with the lipopeptide, the results obtained using various techniques showed that the lipopeptide-based liposomes were well accumulated in cells of the human breast cancer line MDA-MB-231 overexpressing NRP-1. Delivery of reporter genes expressing either β-galactosidase (β-gal) or green fluorescent protein (GFP) was selectively enhanced in these cells when compared with NRP-1-negative cells. In MDA-MB-231 cells, an increase by 250% in β-gal activity was observed when delivered by lipopeptide-based liposomes compared to cationic liposomes alone.

In Vitro and In Vivo Transfection of Melanoma Cells B16-F10 Mediated by Cholesterol-based Cationic Liposomes

In vitro and in vivo transgene expression in B16-F10 melanoma cells has been investigated using an original cationic liposome prepared with triethyl aminopropane carbamoyl cholesterol iodide (TEAPC-Chol) as carrier. TEAPC-Chol/DOPE (dioleoyl phosphatidyl ethanolamine) liposomes are unilamellar, very stable and not toxic in the used concentration range. The yield in complexation with plasmid DNA can reach 100% even in the presence of fetal calf serum. The transfection level has been evaluated by luminometric measurements of luciferase expression. With TEAPC-Chol/DOPE (1:1) liposomes, a relatively high transfection level in B16-F10 cells has been observed comparing to commercial reagents. For in vivo assays, the transfection level in tumors induced in Nude mice has been optimized by studying the effects of charge ratio, of the helper lipid and of the injection volume. Results showed that TEAPC-Chol/DOPE (1:1) liposomes have improved 10-fold transfection level versus direct gene transfer of free DNA.

In vitro and in vivo study of 99mTc-MIBI encapsulated in PEG-liposomes: a promising radiotracer for tumour imaging.

Abstract. Encapsulation of technetium-99m sestamibi (99mTc-MIBI) in polyethyleneglycol-liposomes (99mTc-MIBI-PEG-liposomes) could extend the duration of its circulation in blood and alter its biodistribution, enabling its concentration in tumours to be increased. An original method to encapsulate 99mTc-MIBI in PEG-liposomes is described. The 99mTc-MIBI-PEG-liposomes were compared with free 99mTc-MIBI with respect to (a) tumour availability (b) ability to distinguish between chemotherapy-sensitive and -resistant cells and (c) uptake ratio in tumour imaging. PEG-liposomal systems composed of distearoylphosphatidylcholine/cholesterol/PEG2000-distearoyl phosphatidylethanolamine and lissamine-rhodamine B-labelled liposomes were used. The encapsulation of 99mTc-MIBI in liposomes was achieved using the K+ diffusion potential method. We compared the uptake of free versus encapsulated 99mTc-MIBI by sensitive and resistant erythroleukaemia (K562) and breast tumour (MCF-7ras) cells. To assess the internalisation of these liposomes into cells, rhodamine B-labelled PEG-liposomes were used and visualised by fluorescence microscopy. Biodistribution and imaging characteristics of encapsulated and free radiotracer were determined in rats and tumour-bearing nude mice. The efficiency of 99mTc-MIBI encapsulation in PEG-liposomes was 50±5%. Use of 99mTc-MIBI-PEG-liposomes did not impair the ability of this tracer to distinguish between chemotherapy-sensitive and -resistant tumour cells; the percentage of radioactivity accumulated in the sensitive K562 cells was 1.24±0.04%, as compared with 0.41±0.04% in the resistant K562 cells. One hour post injection in rats, PEG-liposomes showed a ten times higher activity in blood than free 99mTc-MIBI, whereas activity of free 99mTc-MIBI in kidneys and bladder was markedly higher than that of encapsulated 99mTc-MIBI, indicating faster clearance of the free radiotracer. In the (MCF7-ras)-bearing nude mice, PEG-liposome uptake in tumour was two times that of free 99mTc-MIBI. Summarising, the 99mTc-MIBI-PEG-liposomes demonstrated a longer blood circulation time, enabled distinction between chemotherapy-sensitive and -resistant cells and improved tumour to background contrast in in vivo imaging. 99mTc-MIBI-PEG-liposomes therefore show promising potential for tumour imaging.

The interaction of d-propranolol and dimyristoyl phosphatidylcholine large unilamellar vesicles investigated by quasielastic light scattering and Fourier-transform infrared spectroscopy

Abstract The effect of d -propranolol on dimyristoylphosphatidylcholine large unilamellar vesicles has been investigated by quasielastic light scattering and Fourier transform infrared spectroscopy. The results indicate that the drug interacts with the phosphate groups of the polar heads, shifts the phase transition point and alters the low temperature phase. A second transition has been observed, and the new phase of the vesicles induced by the presence of the drug might be an interdigitated one.

Inhibition of VEGF expression in A431 and MDA-MB-231 tumour cells by cationic lipid-mediated siRNA delivery.

In order to promote siRNA transfer in tumour cells, we used an original cationic lipid, synthesized in our laboratory, dimethyl-hydroxyethyl-aminopropane-carbamoyl-cholesterol (DMHAPC-Chol). Liposomes were prepared from this lipid and dioleoylphosphatidylethanolamine (DOPE) in equimolar proportion. Its transfecting capacity was evaluated using ELISA, cell cytometry, and RT-PCR in estimating the silencing effect of VEGF siRNA. This liposome efficiently delivered VEGF siRNA in two human cancer cell lines abundantly secreting VEGF, A431 and MDA-MB-231. Results showed that 50 nM of VEGF siRNA carried by DMHAPC-Chol/DOPE liposomes already silenced more than 90% of VEGF in these cells. A comparative study with two commercial carriers indicated that the inhibition induced by VEGF siRNA transported by cationic DMHAPC-Chol/DOPE liposomes was comparable to that induced by INTERFERin and better than lipofectamine 2000. Moreover, a transfection by a GFP plasmid followed by a GFP siRNA showed that DMHAPC-Chol/DOPE liposomes compared to lipofectamine were less efficient for plasmid but better for siRNA transport. Following one of our previous works concerning cell delivery of plasmid (), the main interest of results presented here resides in the double potential of DMHAPC-Chol/DOPE liposomes to deliver little-sized siRNA as well as large nucleic acids in cells.

Study of emulsions of pharmaceutical interest by light scattering

Abstract Emulsions of light liquid paraffin oil in water have been studied by a light-scattering technique. These emulsions were stabilized with a mixture of simulsol 92 and simulsol 96 as surfactants in the critical HLB range. The influence of the dilution and of the physical parameters of the continuous phase on the translational-diffusion coefficient D a and that of the surfactant HLB, as a function of pH, salinity and temperature, have been established. The extrapolation to infinite dilution of D a leads to an accurate determination of the hydrodynamic radius of the droplets which is in the submicrometre range. On the other hand, the intensity data permitted an estimation of the gyration radius and provided insights into the surfactant layer thickness of the particles. The stability of the emulsions has been discussed in terms of the interaction potential barrier.

Transfer into a mesothelioma cell line of tumor suppressor gene p16 by cholesterol-based cationic lipids.

In this work, the tumor suppressor gene p16 was efficiently transferred into FR cells isolated from a patient with malignant mesothelioma using cationic liposomes prepared from trimethyl aminoethane carbamoyl cholesterol (TMAEC-Chol) and triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol). This transfer was performed after preliminary assays were undertaken to find the optimal transfection conditions. Results showed that an efficient transfer of plasmids containing the reporter gene pCMV-beta galactosidase vectorized by TMAEC-Chol/DOPE and TEAPC-Chol/DOPE liposomes into mesothelioma FR cells was obtained as assessed by luminometric measurements of beta-galactosidase activity. Cytotoxicity studied by MTT test showed that at concentrations used for this study, the cationic liposomes have no effect on cell growth. Transfer into mesothelioma FR cells of a plasmid construct containing the tumor suppressor gene p16 was carried out with these liposomes. Western blotting and immunofluorescence showed the presence of p16 in treated cells. An inhibition of cell growth was observed, indicating that efficient tumor suppressor gene transfer can be performed by using cationic liposomes.