PEI polyplexes

Polyplex surface shielding solves several crucial problems, but may also create new problems. Shielding can strongly reduce the efficiency of subsequent cellular steps of the delivery process [68, 69], and also can negatively alter other polyplex characteristics. For pDNA/PEI polyplexes with optimum medium size of PEI, PEG was found to reduce the polyplex stability in vivo [64, 65, 81]. For a discussion of these aspects see Sect. 3.1. 

A different pH-triggered deshielding concept with hydrophilic polymers is based on reversing noncovalent electrostatic bonds [78, 195, 197]. For example, a pH-responsive sulfonamide/PEl system was developed for tumor-specific pDNA delivery [195]. At pH 7.4, the pH-sensitive diblock copolymer, poly(methacryloyl sulfadimethoxine) (PSD)-hZocA -PEG (PSD-b-PEG), binds to DNA/PEI polyplexes and shields against cell interaction. At pH 6.6 (such as in a hypoxic extracellular tumor environment or in endosomes), PSD-b-PEG becomes uncharged due to sulfonamide protonation and detaches from the nanoparticles, permitting PEI to interact with cells. In this fashion PSD-b-PEG is able to discern the small difference in pH between normal and tumor tissues. 

On the other hand, pDNA/PEI polyplexes were found to be not stable enough in the extracellular in vivo environment. Unpackaging of PEI and PEG-PEI polyplexes was observed [64, 65, 81], for example by serum proteins, soluble glycosaminoglycans, or extracellular matrix components. The situation is even worse in the case of siRNA polyplexes, where PEI polyplexes are dissociated in full human serum, as monitored by fluorescence fluctuation spectroscopy [66, 67]. 

Various researchers have applied the receptor-targeted strategy in pharmacological models for tumor-targeted delivery of pDNA expressing tumor necrosis factor alpha (TNFa). For example, Tf- or Tf-PEG-shielded PEI polyplexes have been used... 

Burke RS, Pun SH (2008) Extracellular barriers to in vivo PEI and PEGylated PEI polyplex-mediated gene delivery to the liver. Bioconjug Chem 19 693-704... 

Fella C, Walker GF, Ogris M, Wagner E (2008) Amine-reactive pyridylhydrazone-based PEG reagents for pH-reversible PEI polyplex shielding. Eur J Pharm Sci 34 309-320... 

Homof M, de la FM, Hallikainen M, Tammi RH, Urtti A (2008) Low molecular weight hyaluronan shielding of DNA/PEI polyplexes facilitates CD44 receptor mediated uptake in human corneal epithelial cells. J Gene Med 10 70-80... 

Boeckle S, Fahrmeir J, Roedl W, Ogris M, Wagner E (2006) Melittin analogs with high lytic activity at endosomal pH enhance transfection with purified targeted PEI polyplexes. J Control Release 112 240-248... 

Fig. 13 Panel for triblock copolymer PEG-PAsp(MPA)-PLL system, a Chemical structure of PEG-PAsp(MPA)-PLL. b Schematic illustration hypothesizing a three-layered micelle formed from the triblock copolymer and pDNA with spatially regulated structure, c In vitro transfection of the luciferase gene to HeLa cells by the micelles from di- or triblock copolymers and polyplex with PEI. The micelles were prepared at a Lys/nucleotide ratio of 2. HeLa cells were incubated with each micelle in a mediiun containing 10% serinn for 24 h, followed by additional 24 h incnbation withont micelles, d The effects of HCQ and NR on the transfection efficiency of the micelles and polyplex. The PEI polyplex was prepared at a N/P ratio of 10. (Fig. 13d Reprinted with permission from [116])...

On the other hand, PEI nanocarriers have some toxicity problems. They are reported to show two types of toxicity one of them is immediate toxicity due to free PEI. Free PEI interacts with serum proteins which have a negative charge and also erythrocytes. This interaction results in precipitation in huge clusters, adherence to the cell membrane and damage to the plasma membrane. The other type is delayed toxicity as a consequence of cellular processing of the PEI polyplexes. Another toxicity problem also arises from the linear structure of PEI. When PEI polyplexes were administered via the intravenous route to mice, lethal side effects were observed. On the other hand, linear PEIs have higher transfection efficiency and lower cytotoxicity than branched PEIs, according to several studies. ... 

PLL can constitute nanosize polyplexes with nucleotides because of its cationic nature. These polyplexes have good cellular uptake when compared with PEI polyplexes, " but PEI polyplexes have a higher transfection efficiency than PLL. PLL polyplexes may be affected by the endosomal acidic pH because of less protonable amine groups compared with To... 

Koping-Hoggard et al. [68] established the relationships between the structure and the properties of chitosan-pDNA polyplexes in vitro and in vivo. They compared polyplexes of ultrapure chitosan (UPC) of preferred molecular structure with those of polyethylenimine (PEI) polyplexes in vitro and after intratracheal administration to mice in vivo. UPC carriers were less cytotoxic than polyethylenimine (PEI) polyplexes and provided a better efficiency compared with that of commonly used cationic lipids. Low-molecular weight chitosan delivery systems were more efficient for cell transfection and less cytotoxic compared with PEL [65]. 

Figure 3.10 Luciferase expression showing the biodistribution 48 h after in vivo administration of PAEP polyplexes with DNA (labelled here as PACN) in mice, their galactose-substituted derivatives (gal-PACN) and PEI polyplexes (PEICN) as a comparison. Reproduced with permission from Y.X. Yang, Z.W. Zhang, L.L. Chen, W.W. Gu and Y.P. Li, Biomacromolecules, 2010, 11, 4, 927. 2010, American Chemical Society [75]...

The type of PEI polymer (molecular weight, branched, or linear form) and the polyplex formulation can largely influence both the toxicity and the gene transfer efficiency [54-56]. For example, purification of PEI polyplexes by size exclusion chromatography [56] recently demonstrated that purified polyplexes, devoid of free PEI, have strongly reduced toxicity both in vitro and in vivo, but also significantly reduced transfection activity free PEI apparently contributes to overcome the bottleneck of endosomal release. 

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]. 

In comparison to PEI polyplexes or standard polylysine polyplexes, dendritic poIy(L-Iysine) KG6 polyplexes appear to have more favorable biophysical characteristics for systemic administration a low zeta potential (+ 3 mV) of particles might be responsible for prolonged circulation in the blood flow. Passive tiunor targeting was observed starting at 1 h after administration. About 1% of the applied DNA dose was observed in the tumor (107]. hi vivo gene expression data have not yet been reported for this system. 

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