Cationic lipid formation

FIG. 15 Cellular entry and intracellular kinetics of the cationic lipid-DNA complexes. Cationic lipid-DOPE liposomes form electrostatic complexes with DNA, and, in this case, also transferrin (Tf) is incorporated. Cellular uptake by endoc5dosis and endosomal acidification can be blocked with cytochaiasin B and bafilomycin Aj, respectively. DNA is proposed to be released at the level of endosomal wall after fusion of the carrier lipids with endosomal bilayer. This process is facilitated by the formation of inverted hexagonal DOPE phase as illustrated in the lower corner on the right. After its release to the C5doplasm DNA may enter the nucleus. (From Ref. 253. By permission of Nature Publishing Group.)... 

Up to 500 pg of plasmid DNA (for the amount of PC shown above) is dissolved in 2mL distilled water, or lOmM sodium phosphate buffer (PB) of pH 7.2 if needed. For liposomes containing both the plasmid DNA and the vaccine protein it encodes (or only the protein), up to 1 mg of the protein is included. The nature of buffer with respect to composition, pH, and molarity can be varied as long as this does not interfere with liposome formation or DNA and protein entrapment yield. Amounts of added DNA and protein can be increased proportionally to the total amount of lipid used. For cationic liposomes, the amount of added DNA can also be increased by employing more cationic lipid. 

Figure 1 The principles and variant parameters of lipofection. (i) Preparation of a lipofection reagent cationic liposomes were prepared from cationic lipids and helper (if required), (ii) Formation of positively charged lipoplexes by addition of DNA (e.g., reporter plasmid carrying the firefly luciferase gene) to the cationic liposomes, (iii) Transfection (lipofection) by incubation cells with the preformed lipoplexes. The efficiency of gene transfer (lipofection efficiency) can be determined from reporter gene amount or activity (e.g., luciferase activity). Most of the steps of a lipofection experiment can be varied and optimized (grey spots).

Methylation of KL-1-14 was an important prerequisite for its transfection properties. A KL-1-14-analog, which was not methylated, did not transfect at all. It could be assumed that the nonmethylated KL-1-14 was not sufficiently protonated at physiological pH, so that the formation of a bilayer structure from these lipids is not possible. As previously shown for DOTAP-analogs, formation of lipid bilayer is an important prerequisites for a cationic lipid to be a transfection lipid (35,47). 

Fig. 10 Schematic illustrations of the formation of a DNA-oriented LB film by using a polyion complex of DNA/intercalator and cationic lipid monolayers...

The necessity of more efficient gene delivery methods prompted the search for novel, less charged or non-cationic gene delivery systems. These non-electrostatic complexes can be advantageous for in vitro and in vivo applications, since unlike cationic lipid/DNA complexes, the novel molecules could not lead to a compacted state of DNA, and could therefore potentially lead to different kinetics of DNA release from complexes. Several compounds are able to bind to double stranded DNA along the grooves by the formation... 

As an alternative to cationic lipids, the potential of anionic lipids for DNA delivery has been investigated. Because of their negative charge, DNA or siRNA molecules are very inefficiently entrapped by anionic lipids alone. In the presence of cations such as K+, Na+, or Ca2+, the complex formation of such anionic lipids and nucleic acids can be enhanced. The resulting ternary complexes of DNA, anionic lipids, and divalent calcium ions have been reported to transfect mammalian cell lines efficiently [33]. Despite this, there has only been limited progress with these anionic lipid DNA delivery systems, a fact that may be attributed, in part, to the poor association between DNA molecules and anionic lipids, caused by electrostatic repulsion between these negatively charged species. 

Holland HE, Shephard L, Sullivan SM (1996) Formation of stable cationic lipid/DNA complexes for gene transfer. Proc Natl Acad Sci USA 93(14) 7305-7309... 

Cationic lipids interact electrostatically and form stable complexes (lipoplexes) with the polyanionic nucleic acids. The structure of most lipoplexes is a multi-lamellar sandwich in which lipid bilayers alternate with layers of DNA strands [16, 62-64] (Fig. 20). Although infrequent, nonlamellar structures have also been found. The free energy gain upon lipoplex formation was shown to be essentially of entropic nature resulting from the counterion release and macromolecule dehydration [65, 66]. 

Lipoplex formation taking place upon DNA mixing with cationic lipid vesicles proceeds in steps of substantially different kinetics (1) adhesion of DNA to the... 

Cellular anionic lipids have a twofold effect on DNA release from the lipo-plexes. They compensate the cationic lipid surface charge and eliminate the electrostatically driven DNA binding to the membrane interface, and they also disrupt the lipoplex structure and facilitate DNA departure into the solution by inducing formation of nonlamellar phases upon mixing with the lipoplex lipids. 

CL-DNA complexes form spontaneously when solutions of cationic liposomes (typically containing both a cationic lipid and a neutral helper lipid) are combined. We have discovered several distinct nanoscale structures of CL-DNA complexes by synchrotron X-ray diffraction, three of which are schematically shown in Fig. 1. These are the prevalent lamellar phase with DNA sandwiched between cationic membranes (Lo,c) [22], the inverted hexagonal phase with DNA encapsulated within inverse lipid tubes (Hnc) [23], and the more recently discovered Hj0 phase with hexagonally arranged rod-like micelles surrounded by DNA chains forming a continuous substructure with honeycomb symmetry [24]. Both the neutral lipid and the cationic lipid can drive the formation of specific structures of CL-DNA complexes. The inverse cone shape of DOPE favors formation of the... 

Hnc phase, while the formation of micelles in the HjC phase is driven by a highly charged (16+), cone-shaped multivalent cationic lipid. 

Boomer, J. A., Thompson, D. H., and Sullivan, S. M. Formation of plasmid-based transfection complexes with an acid-labile cationic lipid Characterization of in vitro and in vivo gene transfer. Pharm Res 19(9) 1292-1301. 2002. 

Liposome-mediated gene delivery is dependent on numerous factors, such as, the formulation of the liposomes including the cationic lipid/neutral lipid ratio, how the liposomes are prepared, the cationic liposome/DNA charge ratio of the complex of cationic liposome and DNA (lipoplex), and the method used to produce the lipoplex. Recently, it was reported that the way in which a liposome was prepared affected transfection efficiency (1), and formation method of lipoplex affected size of lipoplex in which large ones increased the efficiency of transfection (2-7). 

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