Plasmid/cationic carrier complexes

Non-clathrin-coated pit internalization can occur through smooth imagination of 150-300 nm vesicles or via potocytosis. This pathway has been shown to be involved in the transport of folate and other small molecules into the cytoplasm. Plasmids are taken up by muscles through the T-tubules system and caveolae via potocytosis. Muscle cells appear to take up plasmids through the T-tubule system and caveolae via potocytosis. Apart from coated or uncoated pit pathways, cells may also take up plasmid/cationic carrier complexes via plasma membrane destabilization. Particles greater than 200 nm in diameter are not... 

Plasmid/cationic carrier complexes have been proposed to internalize into the endosome and initiate the destabilization of endosomal membranes. This destabilization would induce diffusion of anionic lipids from the external layer of the endosomal membrane into the complexes and form charge neutralized ion pairs with the cationic lipids. Destabilization and/or fusion of the complex with the plasma membrane would permit the same anionic lipids to diffuse to the surface, as would fusion with the endosomal membrane. Release of the condensed DNA from the cationic lipid in the endosome is likely to generate a mechanical or osmotic stress that raptures the endosomal bilayer and releases DNA into the cytoplasm. In contrast, DNA release from complexes on the cell surface might be unable to stress the membrane to a degree sufficient to rapture. 

Plasmid and liposome complexes are easy to produce and are safer, but they have low gene-transfer efficiency. However, novel lipid formulations and synthetic cationic polymer carriers have clearly improved the effectiveness of plasmid-mediated gene transfer [7-9]. 

Cationic peptides have also been used as DNA carriers. For example, gramicidin S and tyrocidine are cationic peptides that will bind to plasmid DNA. When combined with DOPE, the peptide/DNA complex has been shown to transfect cells in vitro. The efficiency of the peptide compared to liposome/ DNA complexes varies by cell type, but the toxicity is equally low (65). Other types of cationic peptides have also been utilized for gene transfer, and they are most effective in combination with molecules that exhibit pH-dependent membrane perturbation effects (30,66). Presumably these helper components promote endocytic escape after cellular uptake. Cationic peptide-type carriers are not in wide usage at this time, particularly in vivo. It will be interesting to see if these peptides induce any immune response when administered to animals. 

In the case of cationic lipid/DNA complexes, the lipid markers that have been used are often the same as those used for conventional liposomes (i.e., CHE). This may not be appropriate. Therefore, we believe it is best to use a radiolabeled form of the lipids used to prepare the carrier system (133). Ideally, carrier cationic lipid and plasmid DNA quantification should be performed on the same tissues within in the same experiment. It may also be useful to analyze the fate of the neutral lipid components of the carrier. Radiolabeled DOPE is available, for example. It may also be useful, in certain types of analyses, such as pharmacokinetic and biodistribution studies, to label both the cationic and neutral components of the lipid carrier. Without some kind of tag, however, the process of detection may be more complex because of the need to efficiently extract the carrier lipid(s) from cells or tissues prior to analysis. The presence of endogenous lipids may make this difficult. For cationic lipids, such as DOTAP or DODAC, for example, it may be recommended that quantification of tissue levels, such as by HPLC analysis, be performed by those specializing in lipid analysis (Northern Lipids, Vancouver, BC, Canada). 

In addition to covalently modified ROMP polymers, noncovalent poly-plexes of cationic ROMP polymers with DNA have been used as transfection agents [218, 219]. Breitenkamp and Emrick showed that PCOE functionalized with cationic polylysine grafts complexed with a plasmid that expresses GFP. This polyplex transfected COS-1 and HeLa cells at comparable or superior levels to commercially available jetPEI (linear polyethyleneimine), SuperFect (dendrimer), and Lipofectamine 2000 (liposomal carrier) transfection vehicles [220]. The ROMP-derived carrier was nontoxic to cells, while all three commercially available reagents led to significant levels of cell death. 

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