One strategy for improving gene vector properties of polyethylenimine is to facilitate individual transfection mechanism steps. This study investigates (i) improving transfection efficiency by attaching peptide nuclear localization signals (nuclear localization signals: SV40 large T antigen nuclear localization signal or C-terminus of histone H1) to polyethylenimine (10 kDa) and (ii) using disulfide linkages, which are expected to be stable during polyplex formation, but cleaved inside cells giving improved gene release. Nuclear localization signal-containing polyplexes exhibited low cytotoxicity, whereas transfection efficiency with high molecular weight plasmid DNA increased up to 3.6 times that of underivatized polyethylenimine in Neuro2A cells at higher molar ratio of polyethylenimine-nitrogen to DNA-phosphate (N/P) ratios. However, with luciferase-specific low molecular weight small interfering RNA in Neuro2A/EGFPLuc cells, nuclear localization signal-containing polyplexes with disulfide linkages caused substantial cytotoxicity at N/P ratios >15 and no consistent significant reduction in luciferase expression. Possible explanations for molecular weight-dependent differences in genetic information transfer by polyplexes containing disulfide-linked nuclear localization signals are discussed.
ForrestMLMeisterGEKoerberJT. Partial acetylation of polyethylenimine enhances in vitro gene delivery. Pharm Res2004; 21: 365–371.
6.
NeuMFischerDKisselT. Recent advances in rational gene transfer vector design based on poly(ethylene imine) and its derivatives. J Gene Med2005; 7: 992–1009.
7.
DemeneixBBehrJBoussifO. Gene transfer with lipospermines and polyethylenimines. Adv Drug Deliv Rev1998; 30: 85–95.
8.
ThomasMKlibanovAM. Enhancing polyethylenimine's delivery of plasmid DNA into mammalian cells. Proc Natl Acad Sci USA2002; 99: 14640–14645.
9.
GabrielsonNPPackDW. Acetylation of polyethylenimine enhances gene delivery via weakened polymer/DNA interactions. Biomacromolecules2006; 7: 2427–2435.
10.
DehshahriAOskueeRKShierWT. Gene transfer efficiency of high primary amine content, hydrophobic, alkyl-oligoamine derivatives of polyethylenimine. Biomaterials2009; 30: 4187–4194.
11.
ShenYPengHDengJ. High mobility group box 1 protein enhances polyethylenimine mediated gene delivery in vitro. Int J Pharm2009; 375: 140–147.
12.
KircheisRKichlerAWallnerG. Coupling of cell-binding ligands to polyethylenimine for targeted gene delivery. Gene Ther1997; 4: 409–418.
13.
ErbacherPRemyJSBehrJP. Gene transfer with synthetic virus-like particles via the integrin-mediated endocytosis pathway. Gene Ther1999; 6: 138–145.
14.
ZantaMABoussifOAdibA. In vitro gene delivery to hepatocytes with galactosylated polyethylenimine. Bioconjug Chem1997; 8: 839–844.
15.
ParkIKLasieneJChouSH. Neuron-specific delivery of nucleic acids mediated by Tet1-modified poly(ethylenimine). J Gene Med2007; 9: 691–702.
LiDWangQQTangGP. Receptor-mediated gene delivery using polyethylenimine (PEI) coupled with polypeptides targeting FGF receptors on cells surface. J Zhejiang Univ Sci B2006; 7: 906–911.
18.
GabrielsonNPPackDW. Efficient polyethylenimine-mediated gene delivery proceeds via a caveolar pathway in HeLa cells. J Control Release2009; 136: 54–61.
19.
KwonEJBergenJMParkIK. Peptide-modified vectors for nucleic acid delivery to neurons. J Control Release2008; 132: 230–235.
20.
TachibanaRHarashimaHShinoharaY. Quantitative studies on the nuclear transport of plasmid DNA and gene expression employing nonviral vectors. Adv Drug Deliv Rev2001; 52: 219–226.
21.
CartierRReszkaR. Utilization of synthetic peptides containing nuclear localization signals for nonviral gene transfer systems. Gene Ther2002; 9: 157–167.
22.
KaouassMBeaulieuRBalickiD. Histonefection: novel and potent non-viral gene delivery. J Control Release2006; 113: 245–254.
23.
ZantaMABelguise-ValladierPBehrJP. Gene delivery: a single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus. Proc Natl Acad Sci USA1999; 96: 91–96.
24.
EguchiAFurusawaHYamamotoA. Optimization of nuclear localization signal for nuclear transport of DNA-encapsulating particles. J Control Release2005; 104: 507–519.
25.
ArisAVillaverdeA. Engineering nuclear localization signals in modular protein vehicles for gene therapy. Biochem Biophys Res Commun2003; 304: 625–631.
26.
BaakeMDoeneckeDAlbigW. Characterisation of nuclear localisation signals of the four human core histones. J Cell Biochem2001; 81: 333–346.
27.
HagstromJESebestyenMGBudkerV. Complexes of non-cationic liposomes and histone H1 mediate efficient transfection of DNA without encapsulation. Biochim Biophys Acta1996; 1284: 47–55.
28.
BottgerMZaitsevSVOttoA. Acid nuclear extracts as mediators of gene transfer and expression. Biochim Biophys Acta1998; 1395: 78–87.
29.
KhadakeJRRaoMR. Condensation of DNA and chromatin by an SPKK-containing octapeptide repeat motif present in the C-terminus of histone H1. Biochemistry1997; 36: 1041–1051.
30.
MoranFMonteroFAzorinF. Condensation of DNA by the C-terminal domain of histone H1. A circular dichroism study. Biophys Chem1985; 22: 125–129.
31.
PueblaIEsseghirSMortlockA. A recombinant H1 histone-based system for efficient delivery of nucleic acids. J Biotechnol2003; 105: 215–226.
32.
TangMXSzokaFC. The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes. Gene Ther1997; 4: 823–832.
33.
BloomfieldVA. Condensation of DNA by multivalent cations: considerations on mechanism. Biopolymers1991; 31: 1471–1481.
34.
MacraeIJZhouKLiF. Structural basis for double-stranded RNA processing by Dicer. Science2006; 311: 195–198.
35.
SnyderSLSobocinskiPZ. An improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines. Anal Biochem1975; 64: 284–288.
36.
BoeckleSFahrmeirJRoedlW. Melittin analogs with high lytic activity at endosomal pH enhance transfection with purified targeted PEI polyplexes. J Control Release2006; 112: 240–248.
37.
MosmannT. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods1983; 65: 55–63.
38.
PanyamJLabhasetwarV. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv Drug Deliv Rev2003; 55: 329–347.
39.
KhalilIAKogureKAkitaH. Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacol Rev2006; 58: 32–45.
40.
OskueeRKDehshahriAShierWT. Alkylcarboxylate grafting to polyethylenimine: a simple approach to producing a DNA nanocarrier with low toxicity. J Gene Med2009; 11: 921–932.
41.
BrandenLJMohamedAJSmithCI. A peptide nucleic acid-nuclear localization signal fusion that mediates nuclear transport of DNA. Nat Biotechnol1999; 17: 784–787.
42.
NiggEA. Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature1997; 386: 779–787.
43.
BauerleMDoeneckeDAlbigW. The requirement of H1 histones for a heterodimeric nuclear import receptor. J Biol Chem2002; 277: 32480–32489.
44.
CarlisleRCBettingerTOgrisM. Adenovirus hexon protein enhances nuclear delivery and increases transgene expression of polyethylenimine/plasmid DNA vectors. Mol Ther2001; 4: 473–483.
45.
BellomoGVairettiMStivalaL. Demonstration of nuclear compartmentalization of glutathione in hepatocytes. Proc Natl Acad Sci USA1992; 89: 4412–4416.
46.
Urban-KleinBWerthSAbuharbeidS. RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo. Gene Ther2005; 12: 461–466.
47.
ReadMLSinghSAhmedZ. A versatile reducible polycation-based system for efficient delivery of a broad range of nucleic acids. Nucleic Acids Res2005; 33: e86–e86.
48.
HassaniZLemkineGFErbacherP. Lipid-mediated siRNA delivery down-regulates exogenous gene expression in the mouse brain at picomolar levels. J Gene Med2005; 7: 198–207.
49.
GraysonACDoodyAMPutnamD. Biophysical and structural characterization of polyethylenimine-mediated siRNA delivery in vitro. Pharm Res2006; 23: 1868–1876.
