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

Kenji Hagiwara, Satoko Kishimoto, Masayuki Ishihara, Yoshiyuki Koyama, Osam Mazda, Toshinori Sato

Research output: Contribution to journalArticle

11 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)83-92
Number of pages10
JournalJournal of Gene Medicine
Volume15
Issue number2
DOIs
Publication statusPublished - 2013 Feb

Fingerprint

Chondroitin Sulfates
Transgenes
Plasmids
Chitosan
Transfection
DNA
Genes
Freeze Drying
Fluid Therapy
Efficiency
Neoplasms
Ganciclovir
Temperature
Aptitude
Thymidine Kinase
Electrophoretic Mobility Shift Assay
Simplexvirus
Luciferases
Genetic Therapy
Suicide

Keywords

  • Anti-tumor effect
  • Chitosan
  • Chondroitin sulfate
  • Freeze-dry
  • Gene transfer
  • Suicide gene

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology
  • Molecular Medicine
  • Genetics(clinical)
  • Drug Discovery

Cite this

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. / Hagiwara, Kenji; Kishimoto, Satoko; Ishihara, Masayuki; Koyama, Yoshiyuki; Mazda, Osam; Sato, Toshinori.

In: Journal of Gene Medicine, Vol. 15, No. 2, 02.2013, p. 83-92.

Research output: Contribution to journalArticle

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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.",
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