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

Sante Di Gioia, Carla Sardo, Giuliana Belgiovine, Daniela Triolo, Maria D'Apolito, Stefano Castellani, Annalucia Carbone, Ida Giardino, Gaetano Giammona, Gennara Cavallaro, Massimo Conese

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Article number14969
Pages (from-to)359-366
Number of pages8
JournalInternational Journal of Pharmaceutics
Volume491
Issue number1-2
DOIs
Publication statusPublished - Jul 13 2015

Keywords

  • Airway epithelial cells
  • HMGB1
  • Nucleic acid-based drugs
  • PHEA
  • Polyaspartamide copolymers
  • Sirna

ASJC Scopus subject areas

  • Pharmaceutical Science

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  • Cite this

    Gioia, S. D., Sardo, C., Belgiovine, G., Triolo, D., D'Apolito, M., Castellani, S., Carbone, A., Giardino, I., Giammona, G., Cavallaro, G., & Conese, M. (2015). Cationic polyaspartamide-based nanocomplexes mediate siRNA entry and down-regulation of the pro-inflammatory mediator high mobility group box 1 in airway epithelial cells. International Journal of Pharmaceutics, 491(1-2), 359-366. [14969]. https://doi.org/10.1016/j.ijpharm.2015.06.017