title: Fabrication and characterization of nuclear localization signal-conjugated glycol chitosan micelles for improving the nuclear delivery of doxorubicin.
abstract: BACKGROUND
  Supramolecular micelles as drug-delivery vehicles are generally unable to enter the nucleus of nondividing cells. In the work reported here, nuclear localization signal (NLS)-modified polymeric micelles were studied with the aim of improving nuclear drug delivery.
  
  
  METHODS
  In this research, cholesterol-modified glycol chitosan (CHGC) was synthesized. NLS-conjugated CHGC (NCHGC) was synthesized and characterized using proton nuclear magnetic resonance spectroscopy, dynamic light scattering, and fluorescence spectroscopy. Doxorubicin (DOX), an anticancer drug with an intracellular site of action in the nucleus, was chosen as a model drug. DOX-loaded micelles were prepared by an emulsion/solvent evaporation method. The cellular uptake of different DOX formulations was analyzed by flow cytometry and confocal laser scanning microscopy. The cytotoxicity of blank micelles, free DOX, and DOX-loaded micelles in vitro was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in HeLa and HepG2 cells.
  
  
  RESULTS
  The degree of substitution was 5.9 cholesterol and 3.8 NLS groups per 100 sugar residues of the NCHGC conjugate. The critical aggregation concentration of the NCHGC micelles in aqueous solution was 0.0209 mg/mL. The DOX-loaded NCHGC (DNCHGC) micelles were observed as being almost spherical in shape under transmission electron microscopy, and the size was determined as 248 nm by dynamic light scattering. The DOX-loading content of the DNCHGC micelles was 10.1%. The DOX-loaded micelles showed slow drug-release behavior within 72 hours in vitro. The DNCHGC micelles exhibited greater cellular uptake and higher amounts of DOX in the nuclei of HeLa cells than free DOX and DOX-loaded CHGC (DCHGC) micelles. The half maximal inhibitory concentration (IC(50)) values of free DOX, DCHGC, and DNCHGC micelles against HepG2 cells were 4.063, 0.591, and 0.171 μg/mL, respectively. Moreover, the IC(50) values of free DOX (3.210 μg/mL) and the DCHGC micelles (1.413 μg/mL) against HeLa cells were nearly 6.96- and 3.07-fold (P < 0.01), respectively, higher than the IC(50) value of the DNCHGC micelles (0.461 μg/mL).
  
  
  CONCLUSIONS
  The results of this study suggest that novel NCHGC micelles could be a potential carrier for nucleus-targeting delivery.
authors.affiliation: Key Laboratory of Systems Biology Medicine of Jiangxi Province, College of Basic Medical Science, Jiujiang University, Jiujiang, China. yjm1016@hotmail.com
authors.forename: Jingmou
authors.initials: J
authors.lastname: Yu
authors.forename: Xin
authors.initials: X
authors.lastname: Xie
authors.forename: Meirong
authors.initials: M
authors.lastname: Zheng
authors.forename: Ling
authors.initials: L
authors.lastname: Yu
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Jianguo
authors.initials: J
authors.lastname: Zhao
authors.forename: Dengzhao
authors.initials: D
authors.lastname: Jiang
authors.forename: Xiangxin
authors.initials: X
authors.lastname: Che
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000008:administration & dosage / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry* / Q000378:metabolism*; D048271:Chitosan / Q000737:chemistry*; D004058:Diffusion; D004317:Doxorubicin / Q000008:administration & dosage* / Q000737:chemistry*; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D008823:Micelles; D053769:Nanocapsules / Q000008:administration & dosage / Q000737:chemistry*; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000493:pharmacokinetics*; D011092:Polyethylene Glycols / Q000737:chemistry
chemical_list: D018906:Antineoplastic Agents, Alkylating; D008823:Micelles; D053769:Nanocapsules; D019913:Nuclear Localization Signals; D011092:Polyethylene Glycols; D004317:Doxorubicin; D048271:Chitosan