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

Abstract

We have reported that polylysine substituted with histidyl residues (His) was 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 determine 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 their transfection efficiency. We found that the presence of free alpha-amino groups allowed the formation of smaller polyplexes but did not modify the zeta potential of +17 mV. At a physiological salt concentration, the adsorption of many serum proteins on His- and AcHis-polyplexes reduced their size below 100 nm, inhibited their aggregation, and reversed their zeta potential 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 polylysine from pH 5.8 to 6.9; in addition, the protonation was further elevated in the presence of pDNA. Serum stabilized negative histidylated polyplexes were less taken up by cells but their transfection efficiency did not decrease; depending on the cell line, His-polyplexes were more efficient than AcHis-polyplexes. The results indicate that (i) the alpha-amino groups of histidyl residues bound to polylysine favorably influence the size and the transfection efficiency of polyplexes, (ii) the alpha-amino groups also elevate the imidazole protonation of His-polyplexes, which is suited to destabilize the membrane of early endocytic vesicles in order to favor pDNA delivery in the cytosol, and (iii) the absorption of selective serum proteins on His-polyplexes could be a way for in vivo gene targeting.

Peptide-guided gene delivery

“…Using polylysine (dp 190), histidine is coupled by reaction with the side-chain amines of lysine to give a partially histidylated polylysine 190 . [78][79][80] Maximal transfection effi ciency was achieved with ~38% of the e -amino groups substituted with histidine residues. 78 Acylation of the a -amino group of the histidyl residues increased the particle size of condensates and decreased transfection effi ciency.…”

“…78 Acylation of the a -amino group of the histidyl residues increased the particle size of condensates and decreased transfection effi ciency. 80 Pichon and coworkers have also shown that a smaller oligolysine (dp 19) can be histidylated, but it was not as effi cient at transfection as the longer polylysine probably because of the inability to tightly compact DNA. 81 Histidylation of low molecular weight disulfi de crosslinking peptides has been demonstrated by both McKenzie et al 21 and Read et al 22 Substitution of histidine for lysine in a cysteine-flanked peptide resulted in short peptides that can bind DNA, have endosomal buffering capacity, and polymerize through disulfi de bonds once condensed with DNA.…”

Transfection efficiency and uptake process of polyplexes in human lung endothelial cells: a comparative study in non‐polarized and polarized cells

“…The size and the charge of the polyplexes were determined using a ZetaSizer 3000 (Malvern Instruments, Orsay, France) as described [24].…”

Cross-linked polyethylenimine–tripolyphosphate nanoparticles for gene delivery

“…These include cationic polymers, which concentrate DNA or RNA into a stable nanoparticle and avoid the problems associated with viral carriers. 11,28,29 Among cationic polymers, such as chitosan, polyamidoamine dendrimers, poly-l-lysine, and poly(2-[dimethylamino] ethyl methacrylate), 30,31 branched PEIs can efficiently link with DNA at different physiological pH values and form PEI-DNA complexes that are diminutive enough to be endocytosed. 32 Although these characteristics of PEI make it an attractive candidate for gene transport, it can cause chargeassociated cytotoxicity at higher concentrations.…”