Role of Polyplex Intermediate Species on Gene Transfer Efficiency: Polyethylenimine−DNA Complexes and Time-Resolved Fluorescence Spectroscopy

Abstract

Polyethylenimine (PEI) is a cationic DNA condensing polymer that facilitates gene transfer into the mammalian cells. The highest gene transfer with branched PEI is obtained at high nitrogen/phosphate (N/P) ratios with free PEI present. The small molecular weight PEI alone is not able to mediate DNA transfection. Here, we used recently developed time-resolved fluorescence spectroscopic method to study the mechanism of PEI-DNA complex formation and to investigate how free PEI, mean molecular weight, and branching of PEI affect the complexes. Analysis of fluorescence lifetimes and time-resolved spectra revealed that for both linear and branched high-molecular-weight PEI the complexation takes place in two steps, but the small-molecular-weight branched PEI complexed DNA at a single step. According to the binding constants obtained from time-resolved spectroscopic measurements, the affinity of N/P complexation per nitrogen atom is highest for LPEI and weakest for BPEI, whereas SPEI-DNA complexation showed intermediate values. Thus, the binding constant alone does not give adequate measure for transfection efficiency. On the other hand, the presence of intermediate states during the polyplex formation seems to be favorable for the gene transfection. Free PEI had no impact on the physical state of PEI-DNA complexes, even though it was essential for gene transfection in the cell culture. In conclusion, the molecular size and topology of PEI have direct influence on the DNA complexation but the free PEI does not. Free PEI must facilitate transfection at the cellular level and not via indirect effects on the PEI-DNA complexes.

Interaction modes between asymmetrically and oppositely charged rods

“…In contrast, the more strongly charged poly(L-lysine) exhibits only a single, tightly bound state with DNA [43]. The presence of a weaker binding state has been suggested [43,44] to be crucial for a polycation to serve as a genetic carrier in gene therapy, as it may enable more effective and controllable release of the DNA from the carrier. This experimental evidence is in line with our results which suggest that this type of polyelectrolyte binding is dependent on charge asymmetry.…”

Electrostatic assembly of a DNA superparamagnetic nano-tool for simultaneous intracellular delivery and in situ monitoring

“…This value is slightly higher than the binding constant for PEI-DNA complexes, which was found to be between 1.10 6 and 2.10 6 M -1 , depending on authors and experimental conditions. 40 Such a constant is compatible with the release of the dODN in the cytoplasm.…”

Independent versus Cooperative Binding in Polyethylenimine–DNA and Poly(l-lysine)–DNA Polyplexes

“…33 This excess polymer has been shown to be a crucial factor in getting DNA to its destination, into the nucleus of the cell. 13,36,51 …”

“…Using time-resolved fluorescence spectroscopy, the binding constants for polyplex formation were determined using an independent binding model. 13 The independent binding model, in contrast to the cooperative binding model, does not take into account the simultaneous or subsequent binding of other amine ligands at unoccupied phosphate sites on DNA. Based on this analysis, we found that for both linear and branched PEI the complexation process was biphasic, suggesting the mechanism of polyplex formation varies as a function of amine concentration.…”

“…In the present study, we continue investigate the mechanism of polyplex formation by comparing the DNA-complexation behavior of branched and linear PEI and PLL at two different pH values in the pH range of endocytic vesicles. The binding equilibrium was analyzed using the independent 13 and cooperative 14 binding models.…”