Lipofection cationic lipids

A promising alternative to viral gene transfer is lipofection, the transfer of the negatively charged DNA material by cationic lipids (13-18). There is no restriction on the size of the therapeutic gene and no risk of immunogeni-city or infection (19). Thus, lipofection in vivo can be principally performed several times (20). Furthermore, cationic lipids can be synthesized in large quantities with relatively little effort. 

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).

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.

Lipofection efficiency by most of the cationic lipids is enhanced by the addition of the neutral lipid, dioleoylphosphatidylethanolamine (DOPE). DOPE promotes the fusion of lipid/DNA particles with the endosomal membrane, inducing their disruption and thus increasing the release of DNA into the cytosol... 

Koynova R, Wang L, MacDonald RC (2007) Synergy in lipofection by cationic lipid mixtures superior activity at the gel-liquid crystalline phase transition. J Phys Chem B 111 7786-7795... 

Li S, Tseng WC, Stolz DB, Wu SP, Watkins SC, Huang L (1999) Dynamic changes in the characteristics of cationic lipidic vectors after exposure to mouse serum implications for intravenous lipofection. Gene Ther 6 585-594... 

Cationic lipid pDNA complexes (lipoplexes) generally are prepared by the simple mixing together of the two components however, it is also important to consider that the applied protocols for complex formation and subsequent modifications strongly influence the properties of the transfection particle. Also, the order of addition of components to form the lipoplex affects considerably lipofection activity. 

---