Role of polyplex intermediate species on gene transfer efficiency: XXXX -DNA complexes and time-resolved fluorescence spectroscopy.

@entity1 @entity1568 @entity12279 @entity1391 @entity8681
@entity12279 ( @entity8681 ) is a cationic DNA condensing polymer that facilitates gene transfer into the @entity1 cells. The highest gene transfer with branched @entity8681 is obtained at high @entity1568 / @entity1391 (N/P) ratios with free @entity8681 present. The small molecular weight @entity8681 alone is not able to mediate DNA transfection. Here, we used recently developed time-resolved fluorescence spectroscopic method to study the mechanism of @entity8681 -DNA complex formation and to investigate how free @entity8681 , mean molecular weight, and branching of @entity8681 affect the complexes. Analysis of fluorescence lifetimes and time-resolved spectra revealed that for both linear and branched high-molecular-weight @entity8681 the complexation takes place in two steps, but the small-molecular-weight branched @entity8681 complexed DNA at a single step. According to the binding constants obtained from time-resolved spectroscopic measurements, the affinity of N/P complexation per @entity1568 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 @entity8681 had no impact on the physical state of @entity8681 -DNA complexes, even though it was essential for gene transfection in the cell culture. In conclusion, the molecular size and topology of @entity8681 have direct influence on the DNA complexation but the free @entity8681 does not. Free @entity8681 must facilitate transfection at the cellular level and not via indirect effects on the @entity8681 -DNA complexes.
