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

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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

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Small Interfering RNA
Down-Regulation
Epithelial Cells
Spermine
poly(2-hydroxyethyl acrylate)
Messenger RNA
Trypan Blue
Electrophoretic Mobility Shift Assay
Oligonucleotides
Flow Cytometry
Polymers
Asthma

Keywords

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

ASJC Scopus subject areas

  • Pharmaceutical Science

Cite this

Cationic polyaspartamide-based nanocomplexes mediate siRNA entry and down-regulation of the pro-inflammatory mediator high mobility group box 1 in airway epithelial cells. / Gioia, Sante Di; Sardo, Carla; Belgiovine, Giuliana; Triolo, Daniela; D'Apolito, Maria; Castellani, Stefano; Carbone, Annalucia; Giardino, Ida; Giammona, Gaetano; Cavallaro, Gennara; Conese, Massimo.

In: International Journal of Pharmaceutics, Vol. 491, No. 1-2, 14969, 13.07.2015, p. 359-366.

Research output: Contribution to journalArticle

Gioia, SD, 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, vol. 491, no. 1-2, 14969, pp. 359-366. https://doi.org/10.1016/j.ijpharm.2015.06.017
Gioia, Sante Di ; Sardo, Carla ; Belgiovine, Giuliana ; Triolo, Daniela ; D'Apolito, Maria ; Castellani, Stefano ; Carbone, Annalucia ; Giardino, Ida ; Giammona, Gaetano ; Cavallaro, Gennara ; Conese, Massimo. / Cationic polyaspartamide-based nanocomplexes mediate siRNA entry and down-regulation of the pro-inflammatory mediator high mobility group box 1 in airway epithelial cells. In: International Journal of Pharmaceutics. 2015 ; Vol. 491, No. 1-2. pp. 359-366.
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T1 - Cationic polyaspartamide-based nanocomplexes mediate siRNA entry and down-regulation of the pro-inflammatory mediator high mobility group box 1 in airway epithelial cells

AU - Gioia, Sante Di

AU - Sardo, Carla

AU - Belgiovine, Giuliana

AU - Triolo, Daniela

AU - D'Apolito, Maria

AU - Castellani, Stefano

AU - Carbone, Annalucia

AU - Giardino, Ida

AU - Giammona, Gaetano

AU - Cavallaro, Gennara

AU - Conese, Massimo

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N2 - 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.

AB - 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.

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KW - HMGB1

KW - Nucleic acid-based drugs

KW - PHEA

KW - Polyaspartamide copolymers

KW - Sirna

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