Lyophilisation and concentration of chitosan/siRNA polyplexes: Influence of buffer composition, oligonucleotide sequence, and hyaluronic acid coating

J Colloid Interface Sci. 2018 Feb 15:512:335-345. doi: 10.1016/j.jcis.2017.09.084. Epub 2017 Sep 22.

Abstract

Chitosan (CS)/siRNA polyplexes have great therapeutic potential for treating multiple diseases by gene silencing. However, clinical application of this technology requires the development of concentrated, hemocompatible, pH neutral formulations for safe and efficient administration. In this study we evaluate physicochemical properties of chitosan polyplexes in various buffers at increasing ionic strengths, to identify conditions for freeze-drying and rehydration at higher doses of uncoated or hyaluronic acid (HA)-coated polyplexes while maintaining physiological compatibility. Optimized formulations are used to evaluate the impact of the siRNA/oligonucleotide sequence on polyplex physicochemical properties, and to measure their in vitro silencing efficiency, cytotoxicity, and hemocompatibility. Specific oligonucleotide sequences influence polyplex physical properties at low N:P ratios, as well as their stability during freeze-drying. Nanoparticles display greater stability for oligodeoxynucleotides ODN vs siRNA; AT-rich vs GC-rich; and overhangs vs blunt ends. Using this knowledge, various CS/siRNA polyplexes are prepared with and without HA coating, freeze-dried and rehydrated at increased concentrations using reduced rehydration volumes. These polyplexes are non-cytotoxic and preserve silencing activity even after rehydration to 20-fold their initial concentration, while HA-coated polyplexes at pH∼7 also displayed increased hemocompatibility. These concentrated formulations represent a critical step towards clinical development of chitosan-based oligonucleotide intravenous delivery systems.

Keywords: Chitosan; Hemocompatibility; Lyophilization/freeze-drying; Nanoparticles; Non-viral gene delivery; siRNA delivery.

MeSH terms

  • Buffers
  • Carcinoma, Non-Small-Cell Lung / drug therapy
  • Carcinoma, Non-Small-Cell Lung / genetics
  • Carcinoma, Non-Small-Cell Lung / pathology*
  • Chitosan / chemistry*
  • Freeze Drying
  • Green Fluorescent Proteins / antagonists & inhibitors*
  • Hemagglutination / drug effects
  • Hemolysis / drug effects
  • Humans
  • Hyaluronic Acid / chemistry*
  • Lung Neoplasms / genetics
  • Lung Neoplasms / pathology
  • Nanoparticles / administration & dosage
  • Nanoparticles / chemistry
  • Oligonucleotides / chemistry*
  • RNA, Small Interfering / administration & dosage*
  • RNA, Small Interfering / chemistry
  • RNA, Small Interfering / genetics
  • Tumor Cells, Cultured

Substances

  • Buffers
  • Oligonucleotides
  • RNA, Small Interfering
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Hyaluronic Acid
  • Chitosan