title: In vivo gene transfer using pDNA/chitosan/chondroitin sulfate ternary complexes: influence of chondroitin sulfate on the stability of freeze-dried complexes and transgene expression in vivo.
abstract: BACKGROUND
  Chitosan has been investigated as a promising nonviral vector. However, several problems still remain, such as a relatively low transfection efficiency and instability under physiological conditions. We previously demonstrated that a chondroitin sulfate (CS) coating enhanced the transfection efficiency and physicochemical stability of plasmid DNA (pDNA)/chitosan complexes in vitro. In the present study, the effects of coating pDNA/chitosan complexes with CS on the stability in freeze-dry rehydration processes and gene expression in vivo were investigated.
  
  
  METHODS
  Freeze-drying storage at -20 °C, 4 °C, or room temperature, freezing storage at -20 °C, or liquid storage at 4 °C or room temperature, were examined for preservation conditions of pDNA/chitosan/CS ternary complexes by a gel retardation assay, measurements of sizes and zeta potentials, and a luciferase assay. Moreover, to determine the transfection efficiency of the ternary complexes in vivo, suicide gene therapy was carried out in Huh-7-implanted mice using herpes simplex virus thymidine kinase coding pDNA and ganciclovir.
  
  
  RESULTS
  The freeze-dried pDNA/chitosan/CS ternary complexes showed sufficient cell transfection ability in vitro and in vivo. In addition, ternary complexes were associated with a significant suppression of tumor growth and a histopathologically high anti-tumor effect by intratumoral injection to tumor-bearing mice.
  
  
  CONCLUSIONS
  The CS coating enhanced the preservation stability of the pDNA/chitosan complexes after freeze-drying-rehydration and their transgene expression in vivo.
authors: Department of Biosciences and Informatics, Keio University, Yokohama, Kanagawa, Japan.
authors: Kenji
authors: K
authors: Hagiwara
authors: Satoko
authors: S
authors: Kishimoto
authors: Masayuki
authors: M
authors: Ishihara
authors: Yoshiyuki
authors: Y
authors: Koyama
authors: Osam
authors: O
authors: Mazda
authors: Toshinori
authors: T
authors: Sato
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D048271:Chitosan / Q000737:chemistry*; D002809:Chondroitin Sulfates / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D005612:Freeze Drying; D015774:Ganciclovir / Q000737:chemistry; D015870:Gene Expression; D018014:Gene Transfer Techniques; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy / Q000379:methods; D006131:Growth Inhibitors / Q000494:pharmacology; D006801:Humans; D015552:Injections, Intralesional; D008156:Luciferases / Q000032:analysis; D008297:Male; D051379:Mice; D010316:Particle Size; D010957:Plasmids / Q000737:chemistry*; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
chemical_list: D000970:Antineoplastic Agents; D006131:Growth Inhibitors; D002809:Chondroitin Sulfates; D004247:DNA; D048271:Chitosan; D008156:Luciferases; D013937:Thymidine Kinase; D001616:beta-Galactosidase; D015774:Ganciclovir