Lipoplexes formation

Gene therapy results to date using this approach have been mixed. The process of lipoplex formation is not easily controlled and hence different batches made under seemingly identical conditions may not be structurally identical. Furthermore, in vitro test results using such lipoplexes can correlate very poorly with subsequent in vivo performance. Clearly, more research is required to underpin the rational use of lipoplexes for gene therapy purposes. The same is true... 

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... 

Fig. 21 Proposed mechanisms of lipoplex formation (a) vesicle titration (DNA initially in excess) -DNA coats the vesicle surfaces as the latter are added to the DNA solution - with increase of the vesicle concentration, clusters of DNA-coated vesicles form and consequently rupture (b) DNA titration (lipid initially in excess) - DNA encounters with bare membranes result in vesicle associations - vesicle-DNA-vesicle adhesion generates stresses, which lead to vesicle rupture, followed by continued aggregation and growth of the complex upon further addition of DNA. (reproduced with permission from [67] copyright (2000) Biophysical Society)...

The SANS data demonstrated that as a result of lipoplex formation, the SUVs were converted to multilamellar lipid-DNA complexes. This transition occurred via three stages. The first step, occurring on a millisecond timescale, was inaccessible to SANS as the smallest time slice used was 1 sec. This step was, however, observable by stopped-flow turbidity and fluorescence experiments. The next step, occurring on a timescale of seconds, which was observable by SANS, was found to correspond to the formation of an (unstable) intermediate with a locally cylindrical structure. The final step, occurring over minutes, involved the conversion of the unstable cylindrical intermediates to a multilamellar structure. As fluorescence measurements can only give information about the conformational changes of DNA, SANS measurements are necessary to probe the structure of the different intermediates that occur during the formation of the DNA-lipid complexes. No other technique lends itself to such studies. 

Oberle V, Bakowsky U, Zuhorn IS, Hoekstra D (2000) Lipoplex formation under equilibrium conditions reveals a three-step mechanism. Biophys J 79 1447-1454... 

Lipoplex Formation Using Liposomes Prepared by Ethanoi injection... 

The molar ratio of cationic liposome to nucleic acid determines the proportion of electrostatic neutralization, which reflects the entire surface charge and the size of resulting lipoplexes (13). Therefore, lipoplex formation should be affected by experimental variables such as nucleic acid/cationic liposome concentration, time and medium for the complexation, the number and/or order of addition steps, and the presence of serum during lipoplex formation. In this section, we will present instructions to form lipoplexes and discuss the most important aspects to be considered in siRNA- or pDNA-lipoplex formation. 

For preparation of lipoplexes, first of all, concentrated stocks of nncleic acid and cationic liposome are prepared and stored at optimal condition (-80°C to 4°C). Then, the concentrated stocks are diluted with required volume of diluent according to desired N/P ratio. An example calculation of N/P ratio is presented. Then, the diluted nucleic acids and liposomes are mixed and stand at room temperature for desired time to allow the lipoplex formation. The formation and stability of complexes can be assessed with the gel retardation assay. 

This is a general procedure for lipoplex formation based on the electrostatic interaction between the positively charged lipid in the liposome and the negatively charged phosphate backbone of the nucleic acid (pDNA or siRNA). 

Combine the diluted nucleic acid solution with the diluted cationic liposome prepared in separated tubes (in the example calculation, the total volume is 1,000 pi). Combine the dilutions in the prescribed order of protocol, since the order of dilution addition is important to achieve the optimal results (See Note 11). Mix by pipetting carefully up and down few times, or by gentle vortexing for 10 s to avoid precipitation, and let it stand for 10-45 min at room temperature to allow the nucleic acid-lipoplex formation. Depending on the concentration of nucleic acid and cationic liposome, the solution may appear cloudy. (See Note 12). 

This method is suggested as an alternative method for siRNA-lipoplex formation, when the spontaneous formation of lipoplex did not produce an effective gene silencing effect (See Note 13). It is important to note that application of vortex-mixing to form pDNA-lipoplex is not practicable, since it may damage the structure of pDNA. 

It is not recommended using media containing serum, antibiotics, or proteins during either dilutions or lipoplex formation, as they may inhibit the complexation process. 

Delivery of recombinant DNA to eukaryotic cells is not a trivial procedure. A method that has been used to help in the transformation includes presenting DNA in the form of complexes (lipoplexes) with cationic lipids, e.g. DOTAP A-([ 1 -(2,3-dioleoyloxy)propyl])-A,A,A-trimethylammonium chloride. Lipoplexes form spontaneously when cationic liposomes and DNA come into contact. One important parameter of lipoplexes, namely hydration, has been measured by D. Hirsch-Lemer and Y. Barenholz using DSC. In this case free water content is computed from the large ice-water transition endotherm at ca. 0 °C. The endotherm for the lipoplex is smaller (as low as 50 per cent in some cases) than the sum of the endotherms for each of the components, showing that dehydration occurs during lipoplex formation. Dehydration happens to be a prerequisite for the intimate contact between cationic lipids and DNA, and is probably instrumental in facilitating DNA transport across the cell membrane. 

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