Cationic polyaspartamide-based nanocomplexes mediate siRNA entry and down-regulation of the pro-inflammatory mediator high mobility group box 1 in airway epithelial cells

Int J Pharm. 2015 Aug 1;491(1-2):359-66. doi: 10.1016/j.ijpharm.2015.06.017. Epub 2015 Jun 30.

Abstract

High-mobility group box 1 (HMGB1) is a nonhistone protein secreted by airway epithelial cells in hyperinflammatory diseases such as asthma. In order to down-regulate HMGB1 expression in airway epithelial cells, siRNA directed against HMGB1 was delivered through nanocomplexes based on a cationic copolymer of poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) by using H441 cells. Two copolymers were used in these experiments bearing respectively spermine side chains (PHEA-Spm) and both spermine and PEG2000 chains (PHEA-PEG-Spm). PHEA-Spm and PHEA-PEG-Spm derivatives complexed dsDNA oligonucleotides with a w/w ratio of 1 and higher as shown by a gel retardation assay. PHEA-Spm and PHEA-PEG-Spm siRNA polyplexes were sized 350-650 nm and 100-400 nm respectively and ranged from negativity/neutrality (at 0.5 ratio) to positivity (at 5 ratio) as ζ potential. Polyplexes formed either at a ratio of 0.5 (partially complexing) or at the ratio of 5 (fully complexing) were tested in subsequent experiments. Epifluorescence revealed that nanocomplexes favored siRNA entry into H441 cells in comparison with naked siRNA. As determined by flow cytometry and a trypan blue assay, PHEA-Spm and PHEA-PEG-Spm allowed siRNA uptake in 42-47% and 30% of cells respectively, however only with PHEA-Spm at w/w ratio of 5 these percentages were significantly higher than those obtained with naked siRNA (20%). Naked siRNA or complexed scrambled siRNA did not exert any effect on HMGB1mRNA levels, whereas PHEA-Spm/siRNA at the w/w ratio of 5 down-regulated HMGB1 mRNA up to 58% of control levels (untransfected cells). PEGylated PHEA-Spm/siRNA nanocomplexes were able to down-regulate HMGB1 mRNA levels up to 61% of control cells. MTT assay revealed excellent biocompatibility of copolymer/siRNA polyplexes with cells. In conclusion, we have found optimal conditions for down-regulation of HMGB1 by siRNA delivery mediated by polyaminoacidic polymers in airway epithelial cells in the absence of cytotoxicity. Functional and in-vivo studies are warranted.

Keywords: Airway epithelial cells; HMGB1; Nucleic acid-based drugs; PHEA; Polyaspartamide copolymers; Sirna.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • DNA / metabolism
  • Down-Regulation / drug effects
  • Epithelial Cells / drug effects
  • Epithelial Cells / metabolism*
  • Gene Transfer Techniques
  • Humans
  • Mammaglobin A / biosynthesis*
  • Materials Testing
  • Nanostructures
  • Peptides / chemistry*
  • Polyhydroxyethyl Methacrylate / analogs & derivatives
  • RNA, Small Interfering / administration & dosage*
  • RNA, Small Interfering / metabolism
  • RNA, Small Interfering / pharmacology*
  • Respiratory Mucosa / cytology
  • Respiratory Mucosa / drug effects
  • Respiratory Mucosa / metabolism
  • Transfection

Substances

  • Mammaglobin A
  • Peptides
  • RNA, Small Interfering
  • SCGB2A2 protein, human
  • Polyhydroxyethyl Methacrylate
  • poly(2-hydroxyethyl acrylate)
  • DNA