Transfection lipofection

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).

Feigner PL, Gadek TR, Holm M, et al. Lipofection a highly efficient, lipid-mediated DNA transfection procedure. ProcNatl Acad Sci USA 1987 84 7413. 

As indicated in Figure 1, the process of lipofection can be divided into independent steps (i) preparation of a lipofection reagent, (ii) formation of lipoplexes, and (iii) the transfection itself. 

For automation, the lipofection process was split of into four independent parts as follows (i) preparation of cationic liposomes, (ii) formation of lipo-plexes, (iii) transfection of the cells, and (iv) quantification of the lipofection efficiency and lipofection-induced cytotoxicity. As shown in Figure 1, this subdivision corresponds to the typical lipofection procedure and each part can be performed separately. 

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.

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. 

To bridge this gap, liposomal transfection efficiency can be dramatically enhanced by the inclusion of peptides into the complex without increasing immunogenicity. Peptides can be selected to assist lipofection at each key stage of the process complex formation, cell targeting and uptake, endosomal disruption, and nuclear targeting. The purpose of this chapter is... 

Peptide modification of liposomes offers the potential for enhancing the packaging process and for enhancing each stage of the lipofection process, to ultimately improve transfection efficiency. 

Figure 1 Potential points for the enhancement of liposome-mediated gene transfer. The above diagram illustrates the characteristic lipofection pathway demonstrating the four key stages bold, underlined), complex formation, targeting and internalization, endosomal escape, and nuclear translocation. Indicated alongside (italic) are the peptides that can be used to augment the transfection potential of the liposome. Abbreviation pDNA, plasmid DNA.

Feigner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M. etal. (1987) Lipofection a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA, 84, 7413-7417. 

In general, transfection methods can be divided into (see Subheading 1.1.1.) physical or direct transfer methods like electroporation, high-velocity bombardment, microinjection and (see Subheading 1.1.2.) chemical methods via carriers, e.g., lipofection, calcium phosphate, and DEAE-dextran. 

Use a different lipofection/transfection method. This may be particularly important when signs of cytotoxicity appear. 

Joshee N, Bastola DR, Cheng PW (2002) Transferrin-facilitated lipofection gene delivery strategy Characterization of the transfection complexes and intracellular trafficking. Hum Gene Ther 13 1991-2004... 

It is interesting also to note that the ability of serum to inhibit lipofection is an often described phenomenon (12). In cell culture systems, liposome-mediated gene transfection is usually carried out in serum-free medium or in at most 10-20% serum. The inhibitory effect of serum on transfection mediated by lipoplexes to hepatocytes (13) has been also reported. Thus, during the in vitro assessment of transfection reagent, it is important to emulate in vivo conditions by using high concentration of serum, as is has been done in this protocol. 

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. 

The transfection efficiency with the gene delivery system by sonoporation mechanism using BLs and US was higher than conventional lipofection method with Lipofectin and Lipofectamine 2000. Therefore, it is expected that this system might be an effective nonviral gene delivery system. 

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