Efficient siRNA delivery and gene silencing using a lipopolypeptide hybrid vector mediated by a caveolae-mediated and temperature-dependent endocytic pathway

Hironori Kasai, Kenji Inoue, Kentaro Imamura, Carlo Yuvienco, Jin Montclare, Seiichi Yamano

Research output: Contribution to journalArticle

Abstract

Background: We developed a non-viral vector, a combination of HIV-1 Tat peptide modified with histidine and cysteine (mTat) and polyethylenimine, jetPEI (PEI), displaying the high efficiency of plasmid DNA transfection with little toxicity. Since the highest efficiency of INTERFERin (INT), a cationic amphiphilic lipid-based reagent, for small interfering RNA (siRNA) transfection among six commercial reagents was shown, we hypothesized that combining mTat/PEI with INT would improve transfection efficiency of siRNA delivery. To elucidate the efficacy of the hybrid vector for siRNA silencing, β-actin expression was measured after siRNA β-actin was transfected with mTat/PEI/INT or other vectors in HSC-3 human oral squamous carcinoma cells. Results: mTat/PEI/INT/siRNA produced significant improvement in transfection efficiency with little cytotoxicity compared to other vectors and achieved ≈ 100% knockdown of β-actin expression compared to non-treated cells. The electric charge of mTat/PEI/INT/siRNA was significantly higher than INT/siRNA. The particle size of mTat/PEI/INT/siRNA was significantly smaller than INT/siRNA. Filipin III and β-cyclodextrin, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI/INT/siRNA transfection, while chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not inhibit mTat/PEI/INT/siRNA transfection. Furthermore, the transfection efficiency of mTat/PEI/INT at 4 °C was significantly lower than 37 °C. Conclusions: These findings demonstrated the feasibility of using mTat/PEI/INT as a potentially attractive non-viral vector for siRNA delivery.

Original languageEnglish (US)
Article number11
JournalJournal of Nanobiotechnology
Volume17
Issue number1
DOIs
StatePublished - Jan 22 2019

Fingerprint

Caveolae
Polyethyleneimine
Gene Silencing
RNA Interference
RNA
Small Interfering RNA
Genes
Temperature
Transfection
Actins
Endocytosis
Filipin
Clathrin
Electric charge
Cyclodextrins
Chlorpromazine
Cytotoxicity
Particle Size
Histidine
Lipids

Keywords

  • Gene delivery
  • Non-viral vector
  • RNA interference
  • Small interfering RNA
  • Transfection

ASJC Scopus subject areas

  • Bioengineering
  • Medicine (miscellaneous)
  • Molecular Medicine
  • Biomedical Engineering
  • Applied Microbiology and Biotechnology
  • Pharmaceutical Science

Cite this

Efficient siRNA delivery and gene silencing using a lipopolypeptide hybrid vector mediated by a caveolae-mediated and temperature-dependent endocytic pathway. / Kasai, Hironori; Inoue, Kenji; Imamura, Kentaro; Yuvienco, Carlo; Montclare, Jin; Yamano, Seiichi.

In: Journal of Nanobiotechnology, Vol. 17, No. 1, 11, 22.01.2019.

Research output: Contribution to journalArticle

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abstract = "Background: We developed a non-viral vector, a combination of HIV-1 Tat peptide modified with histidine and cysteine (mTat) and polyethylenimine, jetPEI (PEI), displaying the high efficiency of plasmid DNA transfection with little toxicity. Since the highest efficiency of INTERFERin (INT), a cationic amphiphilic lipid-based reagent, for small interfering RNA (siRNA) transfection among six commercial reagents was shown, we hypothesized that combining mTat/PEI with INT would improve transfection efficiency of siRNA delivery. To elucidate the efficacy of the hybrid vector for siRNA silencing, β-actin expression was measured after siRNA β-actin was transfected with mTat/PEI/INT or other vectors in HSC-3 human oral squamous carcinoma cells. Results: mTat/PEI/INT/siRNA produced significant improvement in transfection efficiency with little cytotoxicity compared to other vectors and achieved ≈ 100{\%} knockdown of β-actin expression compared to non-treated cells. The electric charge of mTat/PEI/INT/siRNA was significantly higher than INT/siRNA. The particle size of mTat/PEI/INT/siRNA was significantly smaller than INT/siRNA. Filipin III and β-cyclodextrin, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI/INT/siRNA transfection, while chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not inhibit mTat/PEI/INT/siRNA transfection. Furthermore, the transfection efficiency of mTat/PEI/INT at 4 °C was significantly lower than 37 °C. Conclusions: These findings demonstrated the feasibility of using mTat/PEI/INT as a potentially attractive non-viral vector for siRNA delivery.",
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AU - Kasai, Hironori

AU - Inoue, Kenji

AU - Imamura, Kentaro

AU - Yuvienco, Carlo

AU - Montclare, Jin

AU - Yamano, Seiichi

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KW - Transfection

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