Nanoparticle delivery of RNA-based therapeutics to alter the vocal fold tissue response to injury

Nao Hiwatashi, Iv Kraja, Peter A. Benedict, Gregory R. Dion, Renjie Bing, Bernard Rousseau, Milan R. Amin, Danielle M. Nalband, Kent Kirshenbaum, Ryan C. Branski

Research output: Contribution to journalArticle

Abstract

Objectives/Hypothesis: Our laboratory and others hypothesized that Smad3 is a principle mediator of the fibrotic phenotype in the vocal folds (VFs), and we further posited that alteration of Smad3 expression through short interfering (si)RNA holds therapeutic promise, yet delivery remains challenging. To address this issue, we employed a novel synthetic oligomer, lipitoid, complexed with siRNA to improve stability and cellular uptake with the goal of increased efficiency of RNA-based therapeutics. Study Design: In vitro study and in vivo animal model. Methods: In vitro, lipitoid cytotoxicity was quantified via colorimetric and LIVE/DEAD assays in immortalized human VF fibroblasts and primary rabbit VF fibroblasts. In addition, optimal incubation interval and solution for binding siRNA to lipitoid for intracellular delivery were determined. In vivo, a rabbit model of VF injury was employed to evaluate Smad3 knockdown following locally injected lipitoid-complexed siRNA. Results: In vitro, lipitoid did not confer additional toxicity compared to commercially available reagents. In addition, 20-minute incubation in 1× phosphate-buffered saline resulted in maximal Smad3 knockdown. In vivo, Smad3 expression increased following VF injury. This response was significantly reduced in injured VFs at 4 and 24 hours following injection (P = .035 and .034, respectively). Conclusions: The current study is the first to demonstrate targeted gene manipulation in the VFs as well as the potential utility of lipitoid for localized delivery of genetic material in vivo. Ideally, these data will serve as a platform for future investigation regarding the functional implications of therapeutic gene manipulation in the VFs.

Original languageEnglish (US)
JournalLaryngoscope
DOIs
StateAccepted/In press - Jan 1 2017

Fingerprint

Vocal Cords
Nanoparticles
RNA
Wounds and Injuries
Small Interfering RNA
Therapeutics
Fibroblasts
Genes
Rabbits
Animal Models
Phosphates
Phenotype
Injections

Keywords

  • Fibrosis
  • Lipitoid
  • SiRNA
  • Smad3
  • Vocal fold
  • Voice

ASJC Scopus subject areas

  • Otorhinolaryngology

Cite this

Hiwatashi, N., Kraja, I., Benedict, P. A., Dion, G. R., Bing, R., Rousseau, B., ... Branski, R. C. (Accepted/In press). Nanoparticle delivery of RNA-based therapeutics to alter the vocal fold tissue response to injury. Laryngoscope. https://doi.org/10.1002/lary.27047

Nanoparticle delivery of RNA-based therapeutics to alter the vocal fold tissue response to injury. / Hiwatashi, Nao; Kraja, Iv; Benedict, Peter A.; Dion, Gregory R.; Bing, Renjie; Rousseau, Bernard; Amin, Milan R.; Nalband, Danielle M.; Kirshenbaum, Kent; Branski, Ryan C.

In: Laryngoscope, 01.01.2017.

Research output: Contribution to journalArticle

Hiwatashi, N, Kraja, I, Benedict, PA, Dion, GR, Bing, R, Rousseau, B, Amin, MR, Nalband, DM, Kirshenbaum, K & Branski, RC 2017, 'Nanoparticle delivery of RNA-based therapeutics to alter the vocal fold tissue response to injury', Laryngoscope. https://doi.org/10.1002/lary.27047
Hiwatashi, Nao ; Kraja, Iv ; Benedict, Peter A. ; Dion, Gregory R. ; Bing, Renjie ; Rousseau, Bernard ; Amin, Milan R. ; Nalband, Danielle M. ; Kirshenbaum, Kent ; Branski, Ryan C. / Nanoparticle delivery of RNA-based therapeutics to alter the vocal fold tissue response to injury. In: Laryngoscope. 2017.
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AU - Rousseau, Bernard

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AB - Objectives/Hypothesis: Our laboratory and others hypothesized that Smad3 is a principle mediator of the fibrotic phenotype in the vocal folds (VFs), and we further posited that alteration of Smad3 expression through short interfering (si)RNA holds therapeutic promise, yet delivery remains challenging. To address this issue, we employed a novel synthetic oligomer, lipitoid, complexed with siRNA to improve stability and cellular uptake with the goal of increased efficiency of RNA-based therapeutics. Study Design: In vitro study and in vivo animal model. Methods: In vitro, lipitoid cytotoxicity was quantified via colorimetric and LIVE/DEAD assays in immortalized human VF fibroblasts and primary rabbit VF fibroblasts. In addition, optimal incubation interval and solution for binding siRNA to lipitoid for intracellular delivery were determined. In vivo, a rabbit model of VF injury was employed to evaluate Smad3 knockdown following locally injected lipitoid-complexed siRNA. Results: In vitro, lipitoid did not confer additional toxicity compared to commercially available reagents. In addition, 20-minute incubation in 1× phosphate-buffered saline resulted in maximal Smad3 knockdown. In vivo, Smad3 expression increased following VF injury. This response was significantly reduced in injured VFs at 4 and 24 hours following injection (P = .035 and .034, respectively). Conclusions: The current study is the first to demonstrate targeted gene manipulation in the VFs as well as the potential utility of lipitoid for localized delivery of genetic material in vivo. Ideally, these data will serve as a platform for future investigation regarding the functional implications of therapeutic gene manipulation in the VFs.

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