title: Optimize nuclear localization and intra-nucleus disassociation of the exogene for facilitating transfection efficacy of the chitosan.
abstract: Previously, we had reported improving transfection efficiency of the chitosan-plasmid DNA (CS/pDNA)complex via enhancing intracellular unpacking of the exogene by the utilization of phosphorylatable short peptide conjugated chitosan (pSP-CS). In this article, we addressed a novel strategy of nucleus localization signal linked nucleic kinase substrate short peptide (NNS) modification for further optimization of the transfection efficiency. NNS, consisting of "PKKRKVREEAIKFSEEQRFRR", contained a SV40 nucleus localization signal and a potentially phosphorylatable serine residue. The short peptide could be selectively phosphorylated in the nucleus in various mammalian cells. This phosphorylatable NNS (pNNS) was conjugated to chitosan and combined with Cy3 fluorescence labeled plasmid DNA to form a pNNS-CS/pDNA complex. In vitro phosphorylation and DNA releasing assays verified that pNNS could be effectively and selectively phosphorylated by nucleic lysate, hence promoting pDNA unpacking from the complex. Thereafter, C2C12 myoblast cells were transfected. Nuclear localization of the pDNA was represented by the fluorescence in the nucleus and transfection efficiency was determined by the expression of the luciferase reporter gene, which is carried by the plasmid DNA. The results revealed that, compared with lipofactamine2000 and the previously reported pSP-CS, pNNS-CS could transport more pDNA into the nucleus and intensively augment luciferase reporter gene expression. In conclusion, nucleus localization and unpacking from the delivery vector are both critical factors in influencing exogene expression, and pNNS modification is valuable in improving transfection efficacy of the chitosan.
authors.affiliation: Tianjin Medical University, Institute of Endocrinology, Tianjin 300070, China.
authors.forename: Ronglan
authors.initials: R
authors.lastname: Zhao
authors.forename: Bei
authors.initials: B
authors.lastname: Sun
authors.forename: Tieyang
authors.initials: T
authors.lastname: Liu
authors.forename: Yuan
authors.initials: Y
authors.lastname: Liu
authors.forename: Shumin
authors.initials: S
authors.lastname: Zhou
authors.forename: Aijun
authors.initials: A
authors.lastname: Zuo
authors.forename: Dongchun
authors.initials: D
authors.lastname: Liang
mesh_terms: D048271:Chitosan / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004337:Drug Carriers; D004339:Drug Compounding / Q000379:methods*; D016503:Drug Delivery Systems; D017930:Genes, Reporter; D005822:Genetic Vectors; D007202:Indicators and Reagents / Q000378:metabolism; D042581:Intranuclear Space / Q000378:metabolism*; D008055:Lipids / Q000235:genetics / Q000494:pharmacology; D019913:Nuclear Localization Signals / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*
chemical_list: D004337:Drug Carriers; D007202:Indicators and Reagents; D008055:Lipids; C086724:Lipofectamine; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D010455:Peptides; D004247:DNA; D048271:Chitosan