Chitosan-mediated siRNA delivery in vitro: effect of polymer molecular weight, concentration and salt forms

AAPS PharmSciTech. 2010 Mar;11(1):64-72. doi: 10.1208/s12249-009-9355-6. Epub 2010 Jan 8.

Abstract

The aim of this study was to investigate chitosan/siRNA complexes formulated with various chitosan salts (CS) including chitosan aspartate (CS-Asp), chitosan glutamate (CS-Glu), chitosan acetate (CS-Ac), and chitosan hydrochloride (CS-HCl) for in vitro siRNA delivery into stable and constitutive enhanced green fluorescent protein (EGFP)-expressing HeLa cells. The CS/siRNA complexes were characterized by 2% agarose gel electrophoresis and investigated for their transfection efficiency in stable and constitutive EGFP-expressing HeLa cells. The cytotoxicity of the complexes was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The formation of complexes CS/siRNA is mainly dependent on the weight ratio, whereas salt form and molecular weight has less effect. The particle sizes of the complete complexes were in the range of 270-373 nm except the complete complex of CS-Ac, with a slightly positive charge of less than 2 mV. The ability of CS to transfer functionally active siRNA into cell culture is mainly dependent on the weight ratio and molecular weight of CS whereas salt form of CS has less effect. The high gene-silencing efficiency was observed with low MW of CS (20 kDa) and high weight ratio of 32. Over 80% average cell viabilities were observed for CS/siRNA complexes in all weight ratios comparison to untreated cells. This study suggests CS salts have the potential to be used as safe siRNA delivery vectors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Survival / drug effects
  • Cell Survival / genetics
  • Chitosan / metabolism*
  • Chitosan / pharmacology*
  • Cricetinae
  • Electrophoresis, Agar Gel
  • Gene Silencing
  • Genetic Vectors
  • Glutamates / genetics
  • Glutamates / metabolism
  • Glutamates / pharmacology
  • Green Fluorescent Proteins
  • HeLa Cells
  • Humans
  • Molecular Weight
  • Particle Size
  • Polymers / metabolism
  • Polymers / pharmacology
  • RNA, Small Interfering / genetics*
  • RNA, Small Interfering / pharmacology
  • Salts / metabolism
  • Salts / pharmacology*
  • Sodium Chloride / metabolism
  • Sodium Chloride / pharmacology
  • Tetrazolium Salts
  • Thiazoles
  • Transfection

Substances

  • Glutamates
  • Polymers
  • RNA, Small Interfering
  • Salts
  • Tetrazolium Salts
  • Thiazoles
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Sodium Chloride
  • Chitosan
  • thiazolyl blue