Hydrophobic and membrane permeable polyethylenimine nanoparticles efficiently deliver nucleic acids in vitro and in vivo.

Abstract

Conjugation through primary amines is one of the most commonly used methods to modify cationic vectors for efficient gene delivery. Here, dimethyl suberimidate, a commercially available homobifunctional reagent bearing imidoesters at the termini, has been used to crosslink branched polyethylenimine (bPEI) into its nanoparticles (crosslinked PEI nanoparticles, CLP NPs) specifically through primary amines without altering the total charge on the resulting NPs for interaction with biomolecules and cell membranes. By varying the degree of crosslinking, a small series of CLP NPs was prepared and evaluated for their capability to deliver nucleic acids in vitro and in vivo. Physico-chemical characterization revealed the size of the NPs in the range of ∼152 to 210 nm with zeta potential ∼+35 to +38 mV. The plasmid DNA binding ability of these nanoparticles was examined by mobility shift assay, where the pDNA migration was found to be completely retarded by these NPs at an N/P ratio of 4 (cf. bPEI at N/P 3). In various mammalian cells, CLP/pDNA nanoplexes were not only found to be non-toxic but also exhibited significantly enhanced gene expression with one of the formulations, the CLP3/pDNA nanoplex, displaying the highest transfection efficiency, outperforming native bPEI and the selected commercial transfection reagents both in the presence and absence of serum. Further, the versatility of the vector, CLP3, was demonstrated by sequential delivery of GFP-specific siRNA to HEK293 cells, which resulted in ∼79% suppression of the target gene. Intracellular localization studies showed a significant population of the dual labeled nanoplex (CLP3/pDNA) in the nucleus in just 60 min of incubation. Luciferase reporter gene analysis in Balb/c mice post-intravenous administration of the CLP3/pDNA nanoplex showed the highest gene expression in their spleen. The study suggests that CLP NPs could be used as efficient gene delivery vectors for future gene therapy applications.