Lipofectant

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

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. 

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. 

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).

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 3 (A) Robot system for lipofection screening (A) Worktable with racks for microplates, buffer reservoirs, plastic, and glass vials. (B) Four tip liquid handling arm. (C) Gripper for transport of microplates and glass test tubes. (D) High power water bath sonicator. ( ) Nitrogen evaporator. (F) Microplate washer. (G) Absorbance reader. (H) Luminescence reader. (/) Transparent hood. (/) CO2 incubator with pneumatic door (from the rear, front view in B). (B) Self-constructed robotic conveyor for the transport of cell culture plates from the incubator to the worktable.

Screening the Combinatorial Lipid Library for Lipofection Properties... 

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 reagent Mamma carcinoma cells Polarized cells MDCK-C7 Primary cells DC (5 days)... 

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. 

Regelin AE, Fankhaenel S, Gurtesch L, Prinz C, von Kiedrowski G, Massing U. Biophysical and lipofection studies of DOTAP analogs. Biochim Biophys Acta 2000 1464(1) 151-164. 

Opsonization by complement components also represents a potential barrier for intravenous gene delivery. Cationic charges of the particles activate the complement, which then takes part in particle elimination. This hurdle is possibly limited by using short hydrophobic chains, reducing the particle size, and eventually PEG insertion into lipoplexes (18). The interaction effect between the lipoplex and the complement might not be such a limitation. Indeed, it was reported that depletion of complement by injection of cobra venom factor and anti-C3 antibodies in mice indicated no differences upon intravenous injection of lipoplexes, neither in terms of tissue distribution nor in lipofection efficiency (19). 

Simberg D, et al. The role of organ vascularization and lipoplex-serum initial contact in intravenous murine lipofection. J Biol Chem 2003 278 39858. 

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.

Subramanian A, Ma H, Dahl KN, et al. Adenovirus or HA-2 fusogenic pep-tide-assisted lipofection increases cytoplasmic levels of plasmid in nondividing endothelium with little enhancement of transgene expression. J Gene Med 2002 4(l) 75-83. 

Ma H, Zhu J, Maronski M, et al. Non-classical nuclear localization signal peptides for high efficiency lipofection of primary neurons and neuronal cell lines. Neuroscience 2002 112(l) l-5. 

Subramanian A, Ranganathan P, Diamond SL. Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells. Nat Biotechnol 1999 17(9) 873-877. 

Van Tendeloo, V.F., Ponsaerts, P, Lardon, F., et al. (2001). Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood, 98, 49-56. 

For transformation experiments in general, biochemical or physical methods such as microinjection, electroporation or lipofection are available. These methods have been successfully applied for a variety of organisms including protozoan parasites (Clayton, 1999 De Koning-Ward et al., 2000) and were consequently tested for their ability to generate transiently transformed schistosomes by different laboratories. 

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... 

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