Plasmids cellular uptake

Escriou V, Ciolina C, Flelbling-Leclerc A, Wils P, Scherman D. Cationic lipid-mediated gene transfer analysis of cellular uptake and nuclear import of plasmid DNA. Cell Biol Toxicol 1998 14(2) 95-104. 

Spagnou S, Miller AD, Keller M. Lipidic carriers of siRNA differences in the formulation, cellular uptake, and delivery with plasmid DNA. Biochemistry 2004 43(42) 13348-13356. 

Cellular uptake of citric acid is accomplished by the enzyme citrate permease. There is evidence that this enzyme is plasmid-associated in S. lactis subsp. diacetylactis (Cogan 1981 Kempler and McKay 1979). 

By increasing the hydrophobicity of plasmids and reducing their net negative surface charge, PVP may facilitate the uptake of plasmids by muscle cells. Intramuscular injection of PVP-based plasmid formulations in rats significantly increased the number and distribution of expressing cells, as compared to unformulated plasmid. The formation of condensed interpolyelectrolyte complexes between PVP and DNA has been proposed to both protect DNA from nuclease degradation and facilitate its cellular uptake by hydrophobic interaction with cell membranes. The increased hydrophobicity of the complex may enhance interaction with cell membranes and facilitate cell uptake. 

Electrostatic interaction. Cationic CPPs avidly form strong complexes with polyanions and induce the cellular uptake of oligonucleotides (44,45) and plasmids (30,46,47). 

Direct gene transfer into the respiratory system can be carried out for either therapeutic or immunization purposes. Cells in the lung can take up and express plasmid DNA whether it is administered in naked form or formulated with cationic liposomes. For a given dose of DNA, the results can be improved when the DNA is mixed with the minimum amount of lipid that can complex it completely. Such a complex formation can be considered a formation of microparticles that can enhance cellular uptake and subsequent immune responses. 

Figure 14.1. Fate of plasmid DNA in vivo after intravascular (left) or tissue (right) injection. Upon administration, plasmid DNA can be taken up by various cells, including mononuclear phagocytes such as macrophages. It also interacts with plasma proteins and extracellular matrix (ECM) components. In some cases, extravasation (intravascular route) or diffusion (tissue injection) is required for plasmid DNA to reach the target cell. Cellular uptake of DNA occurrs via an endocytotic route (solid lines) as well as a nonendocytotic route (dashed lines), depending on the vector and delivery method used for gene transfer. When endocytosis occurs, a means of endosomal escape is needed. Only plasmid DNA entering the nucleus has a chance to produce therapeutic protein.

Because the size of lipoplex is a key factor in determining the tissue distribution as well as the cellular uptake, it would be a challenge to reduce the size of lipoplex to increase transfection efficiency. Recently, Dauty et al.43 succeeded in formulating plasmid DNA into stable nanometric particles with a diameter of less than 40 nm by synthesizing a dimerizable cationic detergent. 

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. 

Fluorescently labeled plasmid can be quite useful for cellular uptake and distribution studies (112). The main concern with fluorescently tagged DNA is that the presence of the dye molecules may interfere with the interactions between the DNA and the carrier, or the DNA and cellular components. Of equal importance, the binding of the dye to the DNA must be irreversible, so that the dye molecules do not dissociate from the DNA once internalized by the cell. A number of fluorescent DNA-intercalating dyes are available for DNA labeling, such as ethidium bromide, ethidium monoazide, the TO-PRO series, various other cyanine dyes, and many others (82). For example, the membrane-impermeable compound YO-YO... 

Plasmid-based strategies have been developed to transfect cells, hopefully with fewer side effects. In contrast to vimses, plasmids did not evolve to transfer genetic material to mammalian cells, and therefore no active mechanisms of cellular uptake and transport to the nucleus exist. In order to increase the efficacy of gene transfer and protein expression, several modes of packing and transfection have been developed. The... 

For these reasons there were various strategies undertaken to increase its solubility, decrease the toxicity in vitro and in vivo and enhance the cellular uptake of PLL and plasmid DNA (pDNA) complexes. One of the key strategies was to graft PLL with other pol3uners/moieties, which would impart as many aforementioned characteristics as possible without sacrificing its lone virtue, which was to condense DNA via electrostatic interactions. Kim et al. pioneered these developments (22). To repress the expression of glutamic acid... 

We like to emphasize here, that the efficiency of a helper lipid like DOPE is, however, influenced by the packing of the alkyl tails in the membrane formed by the cationic carrier amphiphile. This was demonstrated by a comparative study of two structurally related bilayer-forming surfactants, SAINT-2 (with two C18 l tails) and SAINT-5 (with two C18 0 tails) (Figure 16.10). Both carriers display transfection activity, but DOPE exerts a positive effect on SAINT-2-mediated transfection, but has a negligible effect on transfection mediated by SAINT-5. Interestingly, DOPE effectively enhances DNA dissociation from the lipoplexes formed from both carriers. Most likely, membrane stiffness plays an important role here. Since the bilayer composed of SAINT-5 is more rigid than that formed from SAINT-2, because of the absence of unsaturation in the tails, the plasmid DNA becomes less effectively condensed, and the lipoplex is structurally deformed. This has no effect on cellular uptake but reduces the efficiency of translocation of the plasmid across the membranes of the endosome and/or of the cell nucleus. 

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