In vitro and in vivo gene delivery using chitosan/hyaluronic acid nanoparticles: Influences of molecular mass of hyaluronic acid and lyophilization on transfection efficiency

Toshinori Sato, Mitsuhiro Nakata, Zhihong Yang, Yu Torizuka, Satoko Kishimoto, Masayuki Ishihara

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Background: Lyophilization is an effective method for preserving nonviral gene vectors. To improve the stability and transgene expression of lyophilized plasmid DNA (pDNA) complexes, we coated the surfaces of pDNA/chitosan complexes with hyaluronic acid (HA) of varying molecular masses. The transgene expression of pDNA/chitosan/HA ternary complexes was characterized in vitro and in vivo. Methods: pDNA complexes were lyophilized overnight and the resultant products with spongy, porous consistencies were stored at −30, 4 or 25°C for 2 weeks. Rehydrated complexes were characterized using gel retardation assays, aiming to confirm complex formation, measure particle size and evaluate zeta potential, as well as conduct luciferase gene reporter assays. The anti-tumor effects of pDNA ternary complexes were evaluated using suicide gene (pTK) coding thymidine kinase in Huh7-implanted mice. Results: Transfection efficiencies of pDNA/chitosan/HA ternary complexes were dependent on the average molecular masses of HA. The coating of pDNA/chitosan complexes with HA maintained the cellular transfection efficiencies of lyophilized pDNA ternary complexes. Furthermore, intratumoral injection of lyophilized, rehydrated pDNA ternary complexes into tumor-bearing mice showed a significant suppression of tumor growth. Conclusions: The coating of pDNA/chitosan complexes with high-molecular-weight HA augmented the stability and cellular transfection ability of the complexes after lyophilization-rehydration.

Original languageEnglish
Article numbere2968
JournalJournal of Gene Medicine
Volume19
Issue number8
DOIs
Publication statusPublished - 2017 Aug 1

Fingerprint

Freeze Drying
Chitosan
Hyaluronic Acid
Nanoparticles
Transfection
Plasmids
Efficiency
DNA
Genes
Transgenes
In Vitro Techniques
Neoplasms
Aptitude
Thymidine Kinase
Fluid Therapy
Electrophoretic Mobility Shift Assay
Luciferases
Reporter Genes
Particle Size
Suicide

Keywords

  • anti-tumor effect
  • chitosan
  • gene transfer
  • hyaluronic acid
  • lyophilization
  • suicide gene therapy

ASJC Scopus subject areas

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

Cite this

In vitro and in vivo gene delivery using chitosan/hyaluronic acid nanoparticles : Influences of molecular mass of hyaluronic acid and lyophilization on transfection efficiency. / Sato, Toshinori; Nakata, Mitsuhiro; Yang, Zhihong; Torizuka, Yu; Kishimoto, Satoko; Ishihara, Masayuki.

In: Journal of Gene Medicine, Vol. 19, No. 8, e2968, 01.08.2017.

Research output: Contribution to journalArticle

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abstract = "Background: Lyophilization is an effective method for preserving nonviral gene vectors. To improve the stability and transgene expression of lyophilized plasmid DNA (pDNA) complexes, we coated the surfaces of pDNA/chitosan complexes with hyaluronic acid (HA) of varying molecular masses. The transgene expression of pDNA/chitosan/HA ternary complexes was characterized in vitro and in vivo. Methods: pDNA complexes were lyophilized overnight and the resultant products with spongy, porous consistencies were stored at −30, 4 or 25°C for 2 weeks. Rehydrated complexes were characterized using gel retardation assays, aiming to confirm complex formation, measure particle size and evaluate zeta potential, as well as conduct luciferase gene reporter assays. The anti-tumor effects of pDNA ternary complexes were evaluated using suicide gene (pTK) coding thymidine kinase in Huh7-implanted mice. Results: Transfection efficiencies of pDNA/chitosan/HA ternary complexes were dependent on the average molecular masses of HA. The coating of pDNA/chitosan complexes with HA maintained the cellular transfection efficiencies of lyophilized pDNA ternary complexes. Furthermore, intratumoral injection of lyophilized, rehydrated pDNA ternary complexes into tumor-bearing mice showed a significant suppression of tumor growth. Conclusions: The coating of pDNA/chitosan complexes with high-molecular-weight HA augmented the stability and cellular transfection ability of the complexes after lyophilization-rehydration.",
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