Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung

Abstract

High-molecular-mass polyethylenimines (PEIs) are widely used vectors for nucleic acid delivery. We found that removal of the residual N-acyl moieties from commercial linear 25-kDa PEI enhanced its plasmid DNA delivery efficiency 21 times in vitro, as well as 10,000 times in mice with a concomitant 1,500-fold enhancement in lung specificity. Several additional linear PEIs were synthesized by acid-catalyzed hydrolysis of poly(2-ethyl-2-oxazoline), yielding the pure polycations. PEI87 and PEI217 exhibited the highest efficiency in vitro: 115-fold and 6-fold above those of the commercial and deacylated PEI25s, respectively; moreover, PEI87 delivered DNA to mouse lung as efficiently as the pure PEI25 but at a lower concentration and with a 200-fold lung specificity. These improvements stem from an increase in the number of protonatable nitrogens, which presumably results in a tighter condensation of plasmid DNA and a better endosomal escape of the PEI/DNA complexes. As a validation of the potential of such linear, fully deacylated PEIs in gene therapy for lung diseases, systemic delivery in mice of the complexes of a short interfering RNA (siRNA) against a model gene, firefly luciferase, and PEI25 or PEI87 afforded a 77% and 93% suppression of the gene expression in the lungs, respectively. Furthermore, a polyplex of a siRNA against the influenza viral nucleocapsid protein gene and PEI87 resulted in a 94% drop of virus titers in the lungs of influenza-infected animals.

Fig. 1.

Compound structures and syntheses in this study. (A and B) Chemical structures of aziridine and 2-ethyl-2-oxazoline, respectively. (C) Synthesis of fully deacylated linear PEI25 by exhaustive acid hydrolysis of its commercial predecessor. (D) Synthesis of fully deacylated linear PEI22, PEI87, and PEI217 by the acid hydrolysis of PEOZs. Conditions: (i) 24% (wt/vol) HCl, 110°C, 96 h; k = 517, l = 64, m = 581, PEI25; n = 504 for 50-kDa PEOZ, 2,018 for 200-kDa PEOZ, and 5,044 for 500-kDa PEOZ.

Fig. 2.

Full deacylation increases the buffer capacity and DNA binding efficiency of linear PEI. (A) Acid titration profiles of aqueous solutions of linear commercial PEI25 (squares), fully deacylated PEI25 (open circles), linear PEI87 (filled circles), and NaCl as a control (triangles). Solutions (113 mM) were adjusted to pH 11.5 at room temperature and then titrated with 1 M HCl. (B) Displacement of the intercalated fluorophore EtdBr from plasmid DNA by linear commercial PEI25 (squares), fully deacylated linear PEI25 (open circles), and PEI87 (filled circles).

Fig. 3.

The effect of full deacylation of PEIs on their transfection efficiency and toxicity in vitro. (A) Expression of β-gal in A549 cells after transfection with branched (black bars), commercial linear (red bars), and fully deacylated (green bars) PEI25s, as well as linear, hydrolytically pure PEI22 (yellow bars), PEI87 (blue bars), and PEI217 (pink bars). (B) Cytotoxicities induced by linear polycations: commercial (red circles) and fully deacylated (green triangles) PEI25s, as well as hydrolytically pure PEI22 (yellow circles), PEI87 (blue squares), and PEI217 (pink diamonds) in A549 cells.

Fig. 4.

Comparison of the delivery efficiencies to different organs in mice of a plasmid containing the luciferase gene mediated by different PEIs. Commercial linear (A) and branched (B) PEI25s, as well as hydrolytically pure linear PEI25 (C) and PEI87 (D), were used. Only the mean values are shown.