Histidylated polylysine as DNA vector: Elevation of the imidazole protonation and reduced cellular uptake without change in the polyfection efficiency of serum stabilized negative polyplexes

We have reported that polylysine substituted with histidyl residues (His) w as suited to make complexes with plasmid DNA (pDNA) and to transfect cells in vitro in the presence of serum. The present study was performed to deter mine whether the acetylation of the alpha -amino group of histidyl residues (AcHis) had an influence on the size and the charge of polyplexes and on t heir transfection efficiency. We found that the presence of free alpha -ami no groups allowed the formation of smaller polyplexes but did not modify th e zeta potential of +17 mV. At a physiological salt concentration, the adso rption of many serum proteins on His- and AcHis-polyplexes reduced their si ze below 100 nm, inhibited their aggregation, and reversed their zeta poten tial to -25 mV. The acetylation of the alpha -amino groups reduced slightly the adsorption of serum proteins. The presence of the alpha -amino groups increased the pK of the imidazole protonation of histidine bound to polylys ine from pH 5.8 to 6.9; in addition, the protonation was further elevated i n the presence of pDNA. Serum stabilized negative histidylated polyplexes w ere less taken up by cells but their transfection efficiency did not decrea se; depending on the cell line, His-polyplexes were more efficient than AcH is-polyplexes, The results indicate that (i) the alpha -amino groups of his tidyl residues bound to polylysine favorably influence the size and the tra nsfection efficiency of polyplexes, (ii) the alpha -amino groups also eleva te the imidazole protonation of His-polyplexes, which is suited to destabil ize the membrane of early endocytic vesicles in order to favor pDNA deliver y in the cytosol, and (iii) the absorption of selective serum proteins on H is-polyplexes could be a way for in vivo gene targeting.