Lipid transfection efficiencies

TE of KL-1-14 without helper lipids was very low and reached only about twice the TE, which was found for the standard lipid DOTAP. Independent of the amount of DOPE incorporated in the lipoplexes (ratio of DOPE/KL-1-14 0.3, 0.5, 0.6, 0.7, 0.8, 0.8, 1.0, and 1.2), transfection behaviors (maximum transfection efficiencies and transfection profiles) of all mixtures were similar and comparable to the profile of KL-1-14/DOPE (1 1) as shown in Figure 2 (individual data for all mixtures are not shown). 

Using Choi as helper lipid for KL-1-14, the transfection efficiencies were no longer similar for the different Chol/KL-1-14-ratios (Fig. 7). 

Figure 6 Lipofection results (lipofection profiles) of lipoplexes from the R-configu-rated cationic lipids KL-1-1 to KL-1-17 (Table 1) in a mixture with equimolar amounts of l,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) (counterion chloride) and the pCMVluc-plasmid. Each bar represents the mean ( S.D.) of three wells of a 96-well microtiter plate. T-axis (left) represents the transfection efficiencies expressed in relative light units (RLU) (lu/pg protein). X-axis (right) represents the viability of the cells compared to nontreated control cells. F-axis represents the different cationic lipid/plasmid DNA-charge ratios from 1 to 15.

We compared the TE and toxicity of KL-1-14 synthesized in the R- and S-configuration [with 0.6 mol% Choi as helper lipid (see above)]. The transfection efficiencies for both lipids were statistically similar. Thus, for further experiments, KL-1-14 was synthesized as racemat. 

Transfection efficiencies of the KL-1-14 lipoplexes were compared to the TE achieved with the standard transfection lipid DOTAP. Results were given in RLU (lu/pg protein) and, for easier comparison, standardized on the lipofection efficiency of DOTAP-lipoplexes, which was set to 100% Compared to the respective DOTAP-value. 

Obika S, Yu W, Shimoyama A, et al. Properties of cationic liposomes composed of cationic lipid YKS-220 having an ester linkage adequate stability, high transfection efficiency, and low cytotoxicity. Biol Pharm Bull 1999 22(2) 187-190. 

Numerous studies reported the in vitro interaction of lipoplexes with serum (14). The parameters checked, such as maintained lipid-DNA interactions, aggregate formation, zeta potential changes, or DNA morphological changes, did not allow for consistent prediction of in vitro transfection efficiency of lipoplexes in serum (15). However, it is obviously an indication of poor delivery of DNA at the target site. 

In addition to extracellular degradation in tissues, endosomal acidification might also trigger PEG-lipid cleavage. We showed that despite the presence of the PEG, which slightly reduces lipoplex internalization into the cells, DNA transfection level almost reaches the level of the cationic lipo-plex (31). Cholesterol PEG incorporation into lipoplexes not only reduces lipoplex internalization, but also inhibits the transfection efficiency. 

It was believed that the main factors affecting transfection efficiency were the structure of the cationic lipid, the type of helper lipid used and their susceptibility to disruption by serum proteins. For gene transfer in vivo, apart from DOTMA-based liposomes, other complexes (in equimolar ratios) are also used—such as dioctade-cylamidoglicylspermidin (DLS)/DOPE (137), DOPE/DOTMA (1 1), DOPE/DOTAP (1 1) (138, 139), dimethyloctadecylammonium bromide (DDAB), and DOTAP with cholesterol (1 1) (mol/mol) (139). 

Spermine has been found to enhance the transfection efficiency of DNA-cationic liposome complexes in cell culture and in animal studies this biogenic polyamine at high concentrations caused liposome fusion most likely promoted by the simultaneous interaction of one molecule of spermine (four positively charged amino groups) with the polar head groups of two or more molecules of lipids. At low concentrations (0.03-0.1 mM) it promoted anchorage of the liposome-DNA complex to the surface of cells and enhanced significantly transfection efficiency. 

A number of factors for DOTAP-cholesterol/DNA complex preparation including the DNA/liposome ratio, mild sonication, heating, and extrusion were found to be crucial for improved systemic delivery maximal gene expression was obtained when a homogeneous population of DNA/liposome complexes (200-450 nm) was used. Cryoelectron microscopy showed that the DNA was condensed on the interior of liposomes between two lipid bilayers in these formulations, a factor that was thought to be responsible for the high transfection efficiency in vivo and for the broad tissue distribution (150). 

In a second separate approach we proposed a novel family of lipopolyamines (Byk et al., 1997a,b, 1998a,b,c). The novel approach uses, for the first time, solid phase combinatorial chemistry to obtain a variety of geometrically varied mono-functionalized polyamines. The polyamine, the spacer and the lipid moiety of the lipopolyamines were systematically modified, and the effect of these modifications on transfection efficiency was demonstrated both in vitro and in vivo (Byk et al., 1998a) (Figure 15.8). 

In agreement with the data published by Lee et al. (1996), the geometry of the cationic entity affects the transfection efficiency. Linear-shaped lipopolyamines bearing double lipid chains such as RPR-120535 were more efficient in transfection in various cell lines than T-shaped RPR-126096, globular-shaped... 

Overall, the results obtained for the last two families suggest that the geometry of cationic lipid plays an important role in the final structure of the bilayers and thus affects the transfection efficiency. Nevertheless, this geometric effect seems to differ for different tissue models. 

Unlike DOGSDSO, the present series harbor disulfide bonds in every important position of the cationic lipid. Similarly to RPR120535, these cationic hpids are not formulated with DOPE or other co-lipid(s) for optimal transfection efficiency. Based on exhaustive structure-activity relationship studies, we concluded that RPR-132688 is ten times more active than RPR120535. 

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