PEI/DNA polyplexes

Fig. 6 Endosomal release of PEI/DNA polyplexes. Rupture of a single endosome filled with polyplexes consisting of DNA and PEI labeled by different fluorophores. The upper panel shows the DNA signal and reveals that the intact DNA remains in a confined area of the damaged endosome without diffusion in the cytosol. The lower panel shows the polymer signal whose fluorescence signal disappears due to diffusion into the cytosol. Scale bar 2 pm. Reproduced with permission from Elsevier B.V. [57]...

Generally, DNA complexation to such particles can be performed either by direct crosslinking of PEI/DNA polyplexes with chosen amine-reactive crosslinkers or by complexing the oligonucleotides or DNA by pre-crosslinked PEI particles. It is important to mention that the latter method often yields low molecular weight... 

So far, all the described PEI gene carriers were prepared for ODN or DNA com-plexation by crosslinked PEI particles. Kissel et al. compared the DNA polyplexes prepared either by the method described above or by addition of the DSP crosslinker in DMSO to a buffered aqueous solution of the PEI/DNA polyplexes, and thus by covalent crosslinking after complexation [139], Only the latter method yielded small polyplexes with particle sizes of 100-300 nm, for 0.05 and 0.30 crosslinking degrees, respectively. In addition, pre-crosslinked PEIs showed decreased plasmid compaction and stabilization properties, resulting most probably from incomplete caging of the DNA (Fig. 7). In vivo studies in mice showed that introduction of the crosslinks after polyplex formation makes these polyplexes relatively stable in the circulation, and their transfection efficiency after intravenous administration is retained [140],... 

Recently, van Zanten and coworkers showed that PLL/DNA and PEI/DNA polyplexes maintain their size over long periods of time but decrease in molar mass quickly [121-125], suggesting that liquid storage of these polyplexes may reduce their efficacy. 

Rudolph C, Muller RH, Rosenecker J. Jet nebulization of PEI/DNA polyplexes physical stability and in vitro gene delivery efficiency. J Gene Med, 2002,4(1), 66-74. 

Cell culture experiments [54] showed that large-sized PEI/DNA polyplexes (aggregates of several hundred nm) are more effective but more toxic. The endosomal escape is thought not to be the major bottleneck for such large particles. Small PEI/DNA particles, around 50-100nm, however, have lower efficacy, resulting from an inefficient endosomal release. Photochemical internalization (PCI) is one method to enhance efficiency of the small PEI polyplexes [43,57]. 

Whether the components of the gene carriers actually remain associated during import into the nucleus or enter individually cannot be answered by optical methods as their resolution is limited. A possible technique to study the complexation of DNA within cells is fluorescence correlation spectroscopy (FCS). Clamme et al. studied the intracellular fate of PEI after transfection with polyplexes by two-photon fluorescence FCS [54]. They showed that PEI binds to the inner membrane of endosomes and lysosomes and shows free diffusion in the cytosol as well as the nucleus. However, they did not detect any PEI/DNA complexes inside the nucleus. 

Synthetic polymer. Among the cationic synthetic polymers used for gene delivery are polyethylenimine (PEI), polyamidoamine dendrimers, and poly(2-dimethylaminoethyl methacrylate).161-164 Depending on the flexibility (or rigidity) of the polymers, they form either a small (<100 nm) DNA polyplex or a large (>1 to 10 pm) DNA polyplex.165 More detailed physicochemical properties and their transfection efficacy are to be discussed. 

Conjugation of a DNA/PEI or DNA/PEI-PEG polyplex to targeting moieties such as antibodies to surface antigens like HER2 for breast cancer and OA3 for ovarian cancer has been shown to increase the transfection efficiency in vitro (50,51). PEI can also be lined with amphipathic peptides in order to facilitate cell entry in vivo. PEI coated with the KALA (WEAK-LAKALAKALAKHLAKALAKALKACEA) peptide has been shown to have higher transfection efficiency in a murine model than plain PEI or a commercial liposome (52). 

Enhanced transfection with larger vectors was also seen with lipid-DNA lipoplexes in CHO cells [113]. However, the opposite was found with linear PEI (L-PEI) in vivo. Smaller complexes led to higher levels of gene expression in adult and newborn mice, which correlated to their diffusivity through tissue [100]. In this study, L-PEI DNA complexes were shown to cross the endothelial cell barrier following intravenous administration and preferentially transfect pulmonary cells [92]. Cationic lipids, on the other hand, show some expression in pulmonary cells following intravenous administration but preferentially transfected endothelial cells [51,53,114,115], perhaps due to their reduced stability compared to polyplexes. 

Har-el Y, van Zanten JH, Hanes J. PEI/VEGF DNA polyplexes effects of semm and solvent on vector size, molar mass, and transfection efficiency, in preparation. 

Scheme 20.5 Schematic representations of the preparation of the DNA/PEI complex and the following DNA/PEI/Alginate polyplex. Source From Ref. [97].

DNA polyplexes have been encapsulated and released from polymer microspheres, which may enable these microspheres to be fabricated into matrices using an approach such as the gas-foaming procedure. PLL/DNA complexes have been incorporated into PLG microspheres using a double emulsion process. DNA is incorporated with efficiencies ranging from 30% to 45%, is released over approximately 35 days, and retains its integrity [207, 208]. Alternatively ONs complexed with PEI have been incorporated and released from PLG microspheres. The release profile of the ON/PEI complexes depended on the size, loading, and pore structure of the microspheres. The sustained release of ON/PEI complexes resulted in improved intracellular penetration of the delivered vector as compared with uncom-plexed DNA [209, 210]. PLG/DNA scaffolds have successfully been used in vivo to enhance matrix deposition [113], angiogenesis [112, 113, 115], and bone formation [114]. 

Demeneix B, Behr JP (2005) Polyethylenimine (PEI). Adv Genet 53 217 230 Sonawane ND, Szoka Jr FC, Verkman AS (2003) Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes. J Biol Chem 278 44826 831... 

Owing to their charge, cationic polymers can induce opsonization-mediated aggregation and disassembly of polyplexes in body fluids. Recent in vitro studies have shown PEI-DNA and PLL-DNA polyplexes associate with serum proteins such as albumin, IgM, fibronectin and complement C3, although these unwanted interactions can be negated through modification with hydrophilic polymers. Increase in size in the polyplex systems due to aggregation in systemic circulation could lead to capillaiy bed capture in the... 

Moghimi et al. proposed a two-phase and time-dependent mechanism for the cellular toxicity observed in cell lines after incubation with 25 kDa branched and 750 kDa linear PEI, both in free form and within a DNA polyplex. The first phase typically occurs within 30 min and involves necrotic mechanisms due to plasma membrane permeabilization (Figure 18.4). Arnold et suggested that PLL triggers cellular efflux of organic and inorganic substances proportional to its membrane adsorption. Malik et demonstrated the membrane interactions of polycations... 

The second phase, proposed by Moghimi et al., was said to occur 24 h after exposure, and consisted of a loss of mitochondrial membrane potential which was revealed by translocation of phosphatidylserine as a consequence of PEI-induced channel formation. This led to release of the pro-apoptotic cytochrome c and subsequent activation of caspases-3 triggering apoptosis. Mitochondrial-mediated apoptotic events induced by polycations have also been reported in cell lines treated with high MW PLL in free form, DNA polyplexes and PAMAM dendrimers. In the study by Lee et al., PAMAM dendrimers around 45 nm in size exhibited mitochondrial co-localization, decreased expression of mitochondrial genes and mitochondria membrane... 

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