The possible "proton sponge " effect of polyethylenimine (PEI) does not include change in lysosomal pH.

Polycations such as polyethylenimine (PEI) are used in many novel nonviral vector designs and there are continuous efforts to increase our mechanistic understanding of their interactions with cells. Even so, the mechanism of polyplex escape from the endosomal/lysosomal pathway after internalization is still elusive. The "proton sponge " hypothesis remains the most generally accepted mechanism, although it is heavily debated.

Elucidating the interplay between DNA-condensing and free polycations in gene transfection through a mechanistic study of linear and branched PEI.

In the present study we compare LPEI and BPEI characteristics related to DNA condensation and their role as free polycation chains in gene transfection. Using radioactive (32)P labeled DNA, we investigated the effect of free PEI chains on the cellular uptake of polyplexes. Our investigations show different properties of BPEI and LPEI polyplexes in condensation and de-condensation processes as well as in cellular uptake, which was tightly correlated with transfection efficiency.

Revisit complexation between DNA and polyethylenimine--effect of length of free polycationic chains on gene transfection.

Our revisit of the complexation between DNA and polyethylenimine (PEI) by using a combination of laser light scattering and gel electrophoresis confirms that nearly all the DNA chains are complexed with PEI to form polyplexes when the molar ratio of nitrogen from PEI to phosphate from DNA (N:P) reaches ~3, irrespective of the PEI chain length and solvent. Each solution mixture with N:P>3 contains two kinds of PEI chains: bound to DNA and free in the solution. It has been shown that it is those free PEI chains that play a vital role in promoting the gene transfection.