Cholesterol liposome formation

A study by Bames and co-workers of the equilibrium spreading behavior of dimyristol phosphatidylcholine (DMPC) reconciles the differences between spreading of bulk solids and dispersions of liposomes [41]. This study shows the formation of multibilayers below the monolayer at the air-water interface. An incipient phase separation, undetectable by microscopy, in DMPC-cholesterol... 

Prepare a liposome suspension, containing MPB—PE, at a total lipid concentration of 5 mg/ml in 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.2. Activation of DPPE with SMPB is described in Section 2. A suggested lipid composition for vesicle formation is PC cholesterol PG MPB—PE mixed at a molar ratio of 8 10 1 1. The presence of relatively high levels of cholesterol in the liposomal recipe dramatically enhances the conjugation efficiency of the component MPB—PE groups (Martin et al., 1990). Any method of emulsification to create liposomes of the desired size and morphology may be used (Section 1). 

Waarts BL, Bittman R, WUschut J. SphingoUpid and cholesterol dependence of alphavirus membrane fusion. Lack of correlation with lipid raft formation in target liposomes. J. Biol. Chem. 2002 277 38141-38147. 

Arsonoliposomes (ARSL) which are liposomes that contain arsonolipids in their membranes have shown interesting anticancer and antiparasitic activity in vitro. Their lipid composition (the specific arsonolipids and/or phospholipids used for their preparation, and the relative amounts of each lipid type) highly influences their physicochemical properties as well as their in vivo kinetics and antiparasitic activity however, their cytotoxicity towards cancer cells is minimally - if at all - modified. ARSL are prepared by a modification of the one step method followed or not by sonication (for formation of sonicated or non-sonicated ARSL, respectively). Arsonoliposomes may be composed only of arsonolipids (containing or not cholesterol) [plain ARSL], or they may contain mixtures of arsonolipids with phospholipids (with or without Choi) [mixed ARSL]. Herein, we describe in detail the preparation and physicochemical characterization of ARSL. 

The formation of liposomes [or better arsonoliposomes (ARSL)], composed solely of arsonolipids (Ars with R=lauric acid (C12) myristic acid (C14) palmitic acid (C16) and stearic acid (C18) (Fig. 1) have been used for ARSL construction), mixed or not with cholesterol (Choi) (plain ARSL), or composed of mixtures of Ars and phospholipids (as phosphatidylcholine [PC] or l,2-distearoyl- -glyceroyl-PC [DSPC]) and containing or not Choi (mixed ARSL), was not an easy task. Several liposome preparation techniques (thin-film hydration, sonication, reversed phase evaporation, etc.) were initially tested, but were not successful to form vesicles. Thereby a modification of the so called one step or bubble technique (8), in which the lipids (in powder form) are mixed at high temperature with the aqueous medium, for an extended period of time, was developed. This technique was successfiil for the preparation of arsonoliposomes (plain and mixed) (9). If followed by probe sonication, smaller vesicles (compared to those formed without any sonication [non-sonicated]) could be formed [sonicated ARSL] (9). Additionally, sonicated PEGylated ARSL (ARSL that contain polyethyleneglycol [PEG]-conjugated phospholipids in their lipid bilayers) were prepared by the same modified one-step technique followed by sonication (10). 

Notably, liposomes composed of 3 -[N- N N -dimethylaminoethane)carbamoyl)cholesterol (DC-Chol) together withdioleoylphosphatidylethanolamine (DOPE) (DC-Chol/ DOPE liposome) have been classified as one of the most efficient vectors for the transfection of DNA into cells (8-10) and in clinical trials (11, 12). It has been demonstrated that a 3 2 or 1 1 molar ratio of DC-Chol/DOPE liposome results in high transfection efficiency (10). In these cases, liposomes are mostly prepared by the dry-film method. To further improve the transfection efficiency, it is necessary to evaluate DC-Chol/DOPE liposome from formulation and preparation method of liposome to formation method of their lipoplex. 

Fig. 1. Effect of vortex speed (2,500 rpm for 10 min) on the luciferase gene siiencing efficiency of a siRNA-LipoTrusf lipoplex in HeLa cells. The amount of cationic lipid was 9.6 aM. The siRNA doses correspond to the cationic Npid+/siRNA- charge ratio of 30.45, 15.24,7.62,3.81,1.90 and 0.95, respectively. LipoTrusf is constituted of dioleoylphos-phatidylethanolamine, cholesterol and the cationic lipid 0,0 -ditetradecanoyl-/V-(a-trimethyl ammonioacetyl) diethanolamine chloride (DC-6-14) in the molar ratio of 0.75/0.75/1.00. For this cationic liposome, an expressive gene silencing effect in vitro was obtained at lower siRNA dose with application of a higher vortex-mixing during complex formation...

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