Cationic lipid emulsions containing heavy oils for the transfection of adherent cells
Introduction
Liposomal delivery systems have been extensively studied as potent nonviral gene carriers for several years [1]. Since Straus et al. [2] entrapped adenovirus type 2 DNA in liposomal formulation to transfect KB cells, liposomes have emerged as powerful nonviral vectors. Recently, charged liposomes have been extensively employed to entrap DNA in the inner aqueous phase of liposomes [3], [4]. The synthetic cationic lipid N-[1-(2,3-dioleyloxy)propyl]N,N,N-trimethylammonium chloride (DOTMA) changed this conventional way by introducing the idea of electrostatic complex of DNA/liposome [5]. Therefore, many cationic lipids have been synthesized to develop efficient DNA/lipid complexes, and much effort also has been devoted to find additives for increasing transfection efficiency. For example, a neutral lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was added as a helper lipid into liposome since 1987 [5], [6], [7], [8], [9], [10], [11]. At first, DOPE was introduced as a neutral phospholipid component of the liposome [5]. In this idea, other neutral lipid components such as cholesterol have been used to replace DOPE [6]. DOPE was found to be a helper lipid for cationic lipid-mediated gene transfer because of its fusogenic property facilitating the transfer of DNA from the endosome to the cytosol [7], [8]. Therefore, DOPE has been widely used to formulate many nonviral gene carriers [9], [10], [11].
However, these liposomes also showed some stability problems [12], [13]. Most liposome carriers became unstable in serum proteins, resulting in low transfection efficiencies in the presence of serum [14], [15]. Many alternative cationic nonviral carriers therefore have been developed and some of them are already commercialized for in vitro transfection of DNA, such as TransIT®, Superfect® and FuGene6® [15], [16], [17].
Our previous studies employing cationic lipid emulsions were quite successful in improving their serum stabilities. The lipid emulsions were proven to be good gene delivery systems, especially in the presence of serum and for in vivo transfection [14], [15]. The emulsion, formulated with 1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP)/DOPE/Tween80/squalene, successfully delivered genes for in vitro transfection with serum and for in vivo transfection [16], [17]. The transfection efficiency of the emulsion system was higher than that of the liposomal system in the presence of serum but it was worse than the liposome-based system without serum. The physical properties of the squalene emulsion system were considered as a clue to improve the lower transfection efficiency of the squalene in the absence of serum. In our previous works, squalene showed the most efficient transfection activity even in 80% serum. The emulsion/DNA complex however formed a suspension in the culture medium because the density of squalene was lower than that of water. Due to this property, the emulsion/DNA complex had a low probability to contact the cell surfaces, while the heavier liposome/DNA complex sank to the surface of the adherent cells. DNA/calcium phosphate coprecipitation is a commonly used method for gene transfer. This method uses ‘coprecipitation’ of the DNA and chemicals, resulting in high-density particles. Thus, we could assume that the density should be a crucial factor for the efficient in vitro transfection.
In this work, high-density oil, lipiodol, was employed to form an emulsion for gene carrier. Lipiodol is an iodinated derivative of poppy seed oil, containing ethyl esters of linoleic, oleic, palmitic and stearic acids, with an iodine content of 30–40% (w/v) (as a naturally iodinated compound), and its density is 1.3 (g/ml). In vitro transfection studies under various circumstances were performed to confirm that the density for lipiodol formulations is a key factor for efficient gene transfer in vitro.
Section snippets
Materials
1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were purchased from Avanti Polar Lipids (Alabaster, AL). Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), RPMI 1640 and fetal bovine serum (FBS) were purchased from Invitrogen Life Technologies (Carlsbad, CA). Dimethylsulfoxide (DMSO) and 3-(4,5-dimethylthiazol-3yl)-2,5-diphenyltetrazolum bromide (MTT) were from Sigma-Aldrich (St. Louis, MO). Superfect®
Size and ζ-potential of lipid emulsions and the emulsion/DNA complex
Fig. 1 compares lipiodol emulsions and squalene emulsions, suggesting that the heavy lipiodol emulsion sank to the bottom of the test tube while squalene emulsion with lower density remained in the medium.
The densities of the oil mixtures employing squalene or lipiodol are shown in Table 1. The density of oil mixture was above 1 g/ml when the mixture contained more than 40% (v/v) of lipiodol. The size and the ζ-potential of the emulsions at different oil ratios were measured (Table 1). The
Discussion
In our previous study, cationic lipid emulsions with squalene showed potent transfection efficiency on cultured cells in medium supplemented with a high serum concentration as well as in vivo. At low serum concentration however, it was less effective [16], [17]. This result could be attributed to the fact that the complex of DNA/squalene emulsion formed a suspension and did not precipitate during the transfection, suggesting the complex had a little chance of contacting the cultured cells
Conclusion
We conclude from these results that our new lipiodol emulsion with high density was superior to our previously described squalene emulsion system for in vitro gene transfer of adherent cells. The lipiodol emulsion showed high transfection efficiency and serum stability for in vitro experiments. Therefore, our lipiodol emulsion is expected to be widely used as a potent in vitro transfection agent in the presence of serum.
Acknowledgements
This work was supported by a National Research Laboratory program in Korea.
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