Cationic Cholesterol Research Articles

Synthesis of cholesterol modified cationic lipids for liposomal drug delivery of antisense oligonucleotides

The paper describes a novel synthesis of cholest-5-en-3 β-yl-6-aminohexyl ether (AH-Chol). AH-Chol was used to prepare positively charged liposomes. The liposomes consisted of phospholipon 90H and the cationic cholesterol derivative in an equimolar ratio. Liposome preparation was achieved by membrane homogenization after rehydration of a dry lipid film. Oligonucleotides (ODN) were adsorbed to the cationic liposomes very efficiently. At an ODN/liposome ratio of 1:5 (10:50 μg/ml) 84.2±5.4% of the ODNs were bound to the liposomal membrane. Within the range of 1:40 and 1:100 charge neutralization occurred and the liposome dispersion showed an increase in particle size due to aggregation. Below or above this range of charge neutralization the ODN loaded liposome preparation was physically stable, no sedimentation, increase of vesicle size or vesicle aggregation occurred.

Confocal Laser Scanning Microscopy for Studying Cationic Liposomes Containing a Cationic Cholesterol Derivative with a Hydroxyethylamino Head Group

Confocal Laser Scanning Microscopy for Studying Cationic Liposomes Containing a Cationic Cholesterol Derivative with a Hydroxyethylamino Head Group

Membrane fusion plays an important role in gene transfection mediated by cationic liposomes

By confocal laser scanning microscopy (CLSM) we have studied the membrane fusion between cationic liposomes and the endosome membranes involved in gene transfection mediated by cationic liposomes. Antisense oligonucleotides were transferred by cationic liposomes with a cationic cholesterol derivative, cholesteryl-3β-carboxyamidoethylenedimethylamine ( I). Cationic liposomes were made by a mixture of the derivative I and DOPE. The intracellular distribution of fluorescein-conjugated antisense oligonucleotides (phosphorothioate) was studied by CLSM. The images showed that the antisense oligonucleotides were preferentially transferred into the nucleus of target cells (NIH3T3, COS-7 and HeLa cells) by the liposomes with derivative I. However, their transfection was completely blocked by nigericin which was able to dissipate the pH gradient across the endosome membranes, although the liposome/DNA complex was found in the cytoplasm of the target cells. This was quite in contrast with the fluorescence images of the target cells treated with wortmannin, an inhibitor of endocytosis. The results suggest that at least two steps are effective for gene transfection mediated by the cationic liposomes with cationic cholesterol derivatives. One is the endocytosis of the liposome/DNA complex into the target cells and the other is the removal of antisense oligonucleotides (plasmid DNAs) from the complex in the endosomes. The latter step was preferentially preceded by the membrane fusion between the cationic liposomes and the endosome membranes at around pH 5.0.

Atomic force microscopy for studying gene transfection mediated by cationic liposomes with a cationic cholesterol derivative

Atomic force microscopy (AFM) was used for studying gene transfection mediated by cationic liposomes which contain a cationic cholesterol derivative with a different spacer arm. Cationic liposomes were made by a mixture of one of eight cationic cholesterol derivatives and 1,2-dioleoyl- sn-glycero-3-phosphatidyl ethanolamine (DOPE). AFM images showed that vesicles made of the liposome/DNA complex had various diameters depending on each cationic cholesterol derivative with a different spacer arm. The results showed that the diameter of the liposome/DNA complex was well related to the transfection activity of plasmid pSV2CAT DNA to a cultured cell line (NIH3T3). From the results it was found that the vesicles with moderate diameters (from 0.4 to 1.4 μm) were moste effective for gene transfection of plasmid pSV2CAT DNA into the target cell. Neither smaller vesicles (<400 nm) nor larger vesicles (>1.4 μm) were adequate for gene transfection. As the gene transfection by the cationic liposomes was mostly inhibited by wortmannin, an inhibitor of endocytosis, it is suggested that the vesicles with moderate diameters were useful for gene transfection by endocytosis.

Transfection of Myoblasts in Primary Culture with Isomeric Cationic Cholesterol Derivatives

Transfection of satellite cells from dog muscle (myoblasts) in primary culture has been optimized with respect to the position of the cholesteryl moiety along the polyamine chain of spermidine or spermine. Spermidine or spermine were derivatized with cholesterylchloroformate giving rise to three isomers in the case of spermidine and two isomers for spermine that were separated by reversed-phase high-performance liquid chromatography (rp-HPLC). The position of the cholesteryl moiety was assigned by13C-NMR and coelution with synthetic isomers of defined structure. The isomeric cationic lipids were evaluated for their transfection activity in myoblasts from dog muscle and a human lung epithelial cell line (A549) using plasmid DNA expressing the luciferase reporter gene. The results showed that the position of the cholesteryl moiety is of critical importance for efficient transfection of myoblasts in primary culture with isomers having a derivatized secondary amine being significantly more effective than those with a derivatized primary amine. On the contrary, differences in the A549 cell line were less pronounced and did not follow the same pattern. The results show that slight structural differences between cationic lipids lead to significantly different transfection efficiencies for myoblasts in primary culture. This may also represent an advantage in view of cell or organ targeting.

Cationic cholesterol with a hydroxyethylamino head group promotes significantly liposome-mediated gene transfection

A novel cationic cholesterol derivative with a hydroxyethylamino head group, cholesteryl-3β-carboxyamidoethylene- N-hydroxyethylamine (II), has been synthesized and used for liposome-mediated gene transfection. The cationic liposomes containing the derivative (II) facilitated greatly pSV2CAT gene transfection into mouse NIH3T3 and L929 cells in the absence of serum. The transfection efficiency was much higher than those by the cationic liposomes containing cationic derivatives with a dialkylamino head group (I, III or IV). Further, the efficiency by the cationic liposomes with the derivative (II) was not so much decreased in the presence of serum. This suggested that a novel cationic cholesterol derivative (II) should be very promising in liposome-mediated gene transfection of plasmid and antisense DNA into target cells.

The Interaction of Phospholipid Liposomes with Bacteria and Their Use in the Delivery of Bactericides

Liposomes have been prepared from dipalmitoylphosphatidylcholine (DPPC) incorporating the cationic lipids stearylamine (SA), dimethyldioctadecylammonium bromide (DDAB) and dimethylaminoethane carbamoyl cholesterol (DCchol) and the anionic lipids dipalmitoylphosphatidylglycerol (DPPG) and phosphatidylinositol (PI). Their adsorption to biofilms of skin-associated bacteria (Staphylococcus epidermidis and Proteus vulgaris) and oral bacteria (Streptococcus mutatis and sanguis) has been investigated as a function of mole % cationic and anionic lipid. Targeting (adsorption) was most effective for the systems DPPC-chol-SA, DPPC-DPPG and DPPC-PI liposomes to S. epidermidis. The effect of extracellular mucopolysaccharide on targeting was investigated for S. epidermidis biofilms. It was found that targeting increased with the level of extracellular mucopolysaccharide for all liposome compositions studied. The delivery of the oil-soluble bactericide Triclosan and the water soluble bactericide chlorhexidine was studied for a number of liposomal compositions. Superior delivery of both bactericides relative to the free bactericide occurred for DPPC-chol-SA liposomes and for Triclosan delivery by DPPC-DPPG and DPPC-PI liposomes targeted to S. epidermidis at low bactericide concentrations. DPPC-chol-SA liposomes were also effective for delivery of Triclosan to S. sanguis biofilms. Double labelling experiments using [14C]-chlorhexidine and [3H]-DPPC suggested that there was exchange between adsorbed liposomes which had delivered bactericide to the biofilm and those in the bulk solution implying a diffusion mechanism for bactericide delivery.

Effect of zeta potential of cationic liposomes containing cationic cholesterol derivatives on gene transfection

Cationic liposomes are known to be useful tools for gene transfection. However, the relation between transfection efficiency and physicochemical properties of liposomes has not been well understood. Here, we synthesized eight cationic derivatives of cholesterol which contain a tertiary amino head group with a different spacer arm. Transfection of plasmid pSV2CAT DNA into cells was done by cationic liposomes made of a mixture of dioleoylphosphatidylethanolamine (DOPE) and each cationic cholesterol derivative. At the same time we measured zeta potential of cationic liposomes by laser Doppler spectroscopy. The present results indicated that zeta potentials of cationic liposomes were well related to transfection activity of pSV2CAT DNA. This suggested that zeta potential of cationic liposomes is one of important factors which control gene transfection.

The effect of bilayer composition on calcium ion transport facilitated by fluid shear stress

Passive calcium ion permeability across liposome bilayers is increased during exposure to fluid shear forces attainable in the mammalian vasculature. In this study, liposomes prepared from three different lipid mixtures (phosphatidylcholine alone; phosphatidylcholine and cholesterol; a mixture of anionic and cationic phospholipids plus cholesterol) are exposed to uniform shear stress in a rotational viscometer. Liposome permeability to calcium ion is estimated from continuous measurement of free intraliposome calcium ion concentration using a fluorescence technique. Calcium ion permeability in the absence of fluid force and susceptibility to shear-induced permeability modulation are positively correlated with estimated bilayer compressibility. Fluid shear forces are presumed to influence bilayer packing and modulate defect formation in proportion to bilayer compressibility. Bilayer defects produced by fluid forces may increase liposome permeability.

Cationic liposomes for direct gene transfer in therapy of cancer and other diseases.

Cationic liposomes can mediate efficient delivery of DNA and DNA/protein complex to mammalian cells in vitro and in vivo. Cationic cholesterol derivatives mixed with phosphatidylethanolamine and sonicated to form small unilamellar vesicles can complex with DNA and mediate the entry into the cytosol from the endosome compartment. One of the liposome formulations, DC-Chol liposomes, is used in a gene therapy clinical trial for melanoma. Recently, we exploited these cationic liposomes for the delivery of trans-activating protein factors to regulate and control the expression of delivered transgenes in a protein dose-dependent manner. Bacteriophage T7 RNA polymerase was co-delivered with a reporter gene under the control of T7 promoter to allow cytoplasmic expression of the gene. Human immunodeficiency virus-1 transactivating protein was also codelivered with a reporter gene under the control of HIV-1 long terminal repeat. Finally, human tumor cells selected for cis-platin resistance or isolated from patients who have failed cis-platin therapy are highly transfectable with cationic liposomes. These results suggest a serial therapy protocol with cis-platin and gene therapy for malignancy.

Structure and dynamics of microemulsions. Part 1.—Effect of additives on percolation of conductance and energetics of clustering in water–AOT–heptane microemulsions

Percolation of conductance in the H2O–AOT–heptane system as a function of water content at constant temperature and with variation of temperature at constant composition, in the presence of a large number of additives, are reported. Non-conjugated hydroxy bile salts enhanced percolation whereas cationic surfactants, salts and cholesterol suppressed the process. Various other additives had no effect or assisted the process. The conductance results obeyed the scaling law. Viscosity measurements implied association of microdroplets (cluster formation) during percolation. The energetics of clustering were evaluated by the determination of the dispersion concentration dependent percolation threshold at different temperatures. The enthalpy and entropy of clustering compensate.

Effect of cationic cholesterol derivatives on gene transfer and protein kinase C activity

Four different cationic derivatives of cholesterol were synthesized which contain either a tertiary or a quaternary amino head group, with and without a succinyl spacer-arm. Their ability to inhibit kinase C (PKC) activity was measured in a detergent mixed micellar solution. Derivatives containing a quaternary amino head group were effective inhibitors ( K i approx. 12 and 59 μM) of PKC and derivatives containing a tertiary amino head group were approx. 4–20-fold less inhibitory. Liposomes containing an equimolar mixture of dioleoyphosphatidylethanolamine (DOPE) and a cationic cholesterol derivative were tested for the DNA-mediated transfection activity in mouse L929 cells. Highest activity was found with the derivative with low PKC inhibitory activity and with a succinyl spacer-arm. The transfection activity of this tertiary amine derivative, N, N-dimethylethylenediaminyl succinyl cholesterol was dependent on DOPE as a helper lipid; liposomes containing dioleoylphosphatidylcholine and this derivative had little activity. The transfection protocol of this new cationic liposome reagent was optimized with respect to the ratio of liposome/DNA, dose of the complex and time of incubation with cells. Several adherent cell lines could be efficiently transfected with this liposome reagent without any apparent cytotoxicity. However, the transfection activity was strongly inhibited by the presence of serum components.