Highly effective gene transfection in vivo by alkylated polyethylenimine

Abstract

We mechanistically explored the effect of increased hydrophobicity of the polycation on the efficacy and specificity of gene delivery in mice. N-Alkylated linear PEIs with varying alkyl chain lengths and extent of substitution were synthesized and characterized by biophysical methods. Their in vivo transfection efficiency, specificity, and biodistribution were investigated. N-Ethylation improves the in vivo efficacy of gene expression in the mouse lung 26-fold relative to the parent polycation and more than quadruples the ratio of expression in the lung to that in all other organs. N-Propyl-PEI was the best performer in the liver and heart (581- and 3.5-fold enhancements, resp.) while N-octyl-PEI improved expression in the kidneys over the parent polymer 221-fold. As these enhancements in gene expression occur without changing the plasmid biodistribution, alkylation does not alter the cellular uptake but rather enhances transfection subsequent to cellular uptake.

Figure 1

The effect of N-alkylation on the buffer capacity and DNA binding efficiency of linear PEI. (a) Acid titration profiles of aqueous solutions of the underivatized PEI (solid squares), methyl-PEI (open squares), ethyl-PEI (solid triangles), propyl-PEI (open triangles), butyl-PEI (solid circles), octyl-PEI (open circles), and NaCl as a control (× symbols). The corresponding 113 mM solutions were adjusted to pH 11.5 at room temperature and then titrated with 0.5 M HCl (0.04 M in the case of NaCl). (b) Displacement of the intercalated fluorophore EtBr from plasmid DNA by the underivatized PEI (solid squares), methyl-PEI (open squares), ethyl-PEI (solid triangles), propyl-PEI (open triangles), butyl-PEI (solid circles), and octyl-PEI (open circles). N/P ratio is that between the nitrogen atoms in the polycation and the phosphate groups of the bases in the plasmid.

Figure 2

Comparison of the gene expression in the lungs (a), heart (b), spleen (c), liver (d), and kidneys (e) of a plasmid containing the luciferase gene mediated by the following N-alkylated linear PEI derivatives: (i) unmodified, (ii) methyl, (iii) ethyl, (iv) propyl, (v) butyl, and (vi) octyl.

Figure 3

Gene expression in the lungs of a plasmid containing the luciferase gene mediated by N-ethyl-PEI as a function of the degree of the polycation's alkylation.

Figure 4

(a) Pharmacokinetic profile of ¹²⁵I-labeled gWiz Luc plasmid DNA with no delivery vector with an initial dose of 70 μg of the plasmid containing approximately 1 μCi of radiation. (b) Comparison of the organ biodistribution of ¹²⁵I-labeled plasmid containing the luciferase gene complexed with no vector (black), linear PEI (light grey), and N-ethylated linear PEI (dark grey) at 15 minutes.