title: Efficient intracellular gene delivery using the formulation composed of poly (L-glutamic acid) grafted polyethylenimine and histone.
abstract: OBJECTIVE
  Inefficient endosomal escape and poor nuclear import are thought to contribute to low gene transfer efficiency of polycations. To overcome these drawbacks, we prepared multiple gene delivery formulations including low cytotoxic polycation, histone containing NLSs and chloroquine as the endosomolytic agent.
  
  
  METHODS
  Comb-shaped poly (L-glutamic acid) grafted low-molecular-weight polyethylenimine (PLGE) copolymer was synthesized by aminolysis of poly-γ-benzyl-L-glutamate using low-molecular-weight polyethylenimine (800 Da). The formation of DNA/histone/PLGE terplex was observed by atomic force microscope and gel retardation assay. The particle size and zeta potential of DNA complexes with varying content of histone were also measured to confirm the terplex formation. Cytotoxicity of vectors was assayed by MTT. Multiple gene delivery formulations were optimized to their best transfection efficiency that was monitored by fluorescence microscope and flow cytometry. In vivo gene delivery of the optimal formulation was evaluated by the GFP-expression levels in drosophila melanogaster.
  
  
  RESULTS
  The DNA/histone/PLGE terplex was successfully formed. The PLGE and histone together condensed DNA into small, discrete particles (less than 200 nm in diameter) in isotonic solution. Cytotoxicity of PLGE and histone were much lower than that of PEI 25 K. Either histone or chloroquine contributed to enhancing the levels of transfection activity of PLGE polymer. However, chloroquine and histone did not show a synergistic effect on the improvement of transfection efficiency. The optimal formulation was the DNA/histone/PLGE terplex at the N/P ratio of 15 and histone/ DNA weight ratio of 0.8. Compared with Lipofectamine 2000 and PEI 25 K, the optimal formulation showed significantly increased levels of GFP-expression both in vitro and in vivo.
  
  
  CONCLUSIONS
  This formulation provided a versatile approach for preparing high efficiency of the polycation-based gene vectors. It also reinforced the finding of earlier studies that nuclear import and endosomal escape were rate-limiting steps for nonviral gene delivery.
authors.affiliation: Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, Guangzhou, 510006, People's Republic of China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Deng
authors.forename: Yuting
authors.initials: Y
authors.lastname: Wen
authors.forename: Cuifeng
authors.initials: C
authors.lastname: Wang
authors.forename: Shirong
authors.initials: S
authors.lastname: Pan
authors.forename: Huaiyu
authors.initials: H
authors.lastname: Gu
authors.forename: Xin
authors.initials: X
authors.lastname: Zeng
authors.forename: Lina
authors.initials: L
authors.lastname: Han
authors.forename: Yuefang
authors.initials: Y
authors.lastname: Zhao
authors.forename: Min
authors.initials: M
authors.lastname: Feng
authors.forename: Chuanbin
authors.initials: C
authors.lastname: Wu
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002738:Chloroquine / Q000737:chemistry; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics; D004331:Drosophila melanogaster / Q000235:genetics; D004337:Drug Carriers / Q000009:adverse effects / Q000138:chemical synthesis / Q000737:chemistry*; D011992:Endosomes / Q000378:metabolism; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry*; D006801:Humans; D015394:Molecular Structure; D008970:Molecular Weight; D019913:Nuclear Localization Signals / Q000737:chemistry; D010316:Particle Size; D010957:Plasmids; D011094:Polyethyleneimine / Q000009:adverse effects / Q000138:chemical synthesis / Q000737:chemistry*; D011099:Polyglutamic Acid / Q000009:adverse effects / Q000138:chemical synthesis / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013499:Surface Properties; D014162:Transfection
chemical_list: D004337:Drug Carriers; D006657:Histones; D019913:Nuclear Localization Signals; C559740:poly (L-glutamic acid) grafted polyethylenimine; D049452:Green Fluorescent Proteins; D011099:Polyglutamic Acid; D002738:Chloroquine; D011094:Polyethyleneimine; D004247:DNA