Lipoplex formation, result

Independent of the nature of serum protein absorbed on the lipoplexes, the resulting effect is the formation of large aggregates, inducing... 

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]. 

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. 

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. 

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). 

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. 

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