Multifunctional nanocarrier based on clay nanotubes for efficient intracellular siRNA delivery and gene silencing

Abstract

RNA interference-mediated gene silencing relating to disease has recently emerged as a powerful method in gene therapy. Despite the promises, effective transport of siRNA with minimal side effects remains a challenge. Halloysites are cheap and naturally available aluminosilicate clay nanotubes with high mechanical strength and biocompatibility. In this study, a novel multifunctional nanocarrier based on functionalized halloysite nanotubes (f-HNTs) has been developed via electrostatic layer-by-layer assembling approach for loading and intracellular delivery of therapeutic antisurvivin siRNA and simultaneously tracking their intracellular transport, in which PEI-modified HNTs are used as gene vector, antisurvivin siRNA as gene therapeutic agent, and mercaptoacetic acid-capped CdSe quantum dots as fluorescent labeling probes. The successful assembly of the f-HNTs-siRNA complexes was systematically characterized by transmission electron microscopy (TEM), UV–visible spectrophotometry, Zeta potential measurement, fluorescence spectrophotometry, and electrochemical impedance spectroscopy. Confocal microscopy, biological TEM, and flow cytometry studies revealed that the complexes enabled the efficient intracellular delivery of siRNA for cell-specific gene silencing. MTT assays exhibited that the complexes can enhance antitumor activity. Furthermore, Western blot analysis showed that f-HNTs-mediated siRNA delivery effectively knocked down gene expression of survivin and thereby decreased the levels of target proteins of PANC-1 cells. Therefore, this study suggested that the synthesized f-HNTs were a new effective drug delivery system for potential application in cancer gene therapy.

Keywords

Multifunctional nanocarrier halloysite nanotubes siRNA intracellular delivery gene silencing

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References

Altieri

. Survivin, versatile modulation of cell division and apoptosis in cancer. Oncogene 2003; 22: 8581–8589.

Altieri

. Validating survivin as a cancer therapeutic target. Nat Rev Cancer 2003; 3: 46–54.

Beltrami

Plescia

Wilkinson

. Acute ablation of survivin uncovers p53-dependent mitotic checkpoint functions and control of mitochondrial apoptosis. J Biol Chem 2004; 279: 2077–2084.

Boussif

Lezoualc’h

Zanta

. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci 1995; 92: 7297–7301.

Dohi

Beltrami

Wall

. Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis. J Clin Invest 2004; 114: 1117–1127.

Fesik

. Promoting apoptosis as a strategy for cancer drug discovery. Nat Rev Cancer 2005; 5: 876–885.

Fire

Montgomery

. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998; 391: 806–811.

Guimaraes

Enyashin

Seifert

. Structural, electronic, mechanical properties of single-walled halloysite nanotube models. J Phys Chem C 2010; 114: 11358–11363.

Herrero

Toma

Al-Jama

. Synthesis and characterization of a carbon nanotube-dendron series for efficient siRNA delivery. J Am Chem Soc 2009; 131: 9843–9848.

10.

Hoffman

Biade

Zilfou

. Transcriptional repression of the anti-apoptotic survivin gene by wild type p53. J Biol Chem 2002; 277: 3247–3257.

11.

Jia

Lian

Shen

. Intracellular delivery of quantum dots tagged antisense oligodeoxynucleotides by functionalized multiwalled carbon nanotubes. Nano Lett 2007; 7: 2976–2980.

12.

Kim

Jang

Ryou

. A functionalized gold nanoparticles-assisted universal carrier for antisense DNA. Chem Commun 2010; 46: 4151–4153.

13.

Kim

Hou

. Opsonized erythrocyte ghosts for liver-targeted delivery of antisense oligodeoxynucleotides. Biomaterials 2009; 30: 959–967.

14.

Lee

Williams

Devore

. Poly(propylacrylic acid) enhances cationic lipid-mediated delivery of antisense oligonucleotides. Biomacromolecules 2006; 7: 1502–1508.

15.

Lee

Yoon

Lee

. Direct 3-D imaging of the evolution of block copolymer microstructures using laser scanning confocal microscopy. Macromolecules 2007; 40: 6021–6024.

16.

Ackermann

Bennett

. Pleiotropic cell-division defects and apoptosis induced by interference with survivin function. Nat Cell Biol 1999; 1: 461–466.

17.

Nie

Zhang

. Investigation of PEG-PLGA-PEG nanoparticles-based multipolyplexes for IL-18 gene delivery. J Biomater Appl 2012; 26: 893–916.

18.

Ambrosini

Chu

. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 1998; 396: 580–584.

19.

Huang

. Targeted delivery of antisense oligodeoxynucleotide and small interference RNA into lung cancer cells. Mol Pharm 2006; 3: 579–588.

20.

Liu

Xie

Zhang

. N-Alkyl-PEI-functionalized iron oxide nanoclusters for efficient siRNA delivery. Small 2011; 7: 2742–2749.

21.

Liu

Biddle

Hanks

. Activation of dual apoptotic pathways in human melanocytes and protection by survivin. J Invest Dermatol 2006; 126: 2247–2256.

22.

Liu

Franzen

. Factors determining the efficacy of nuclear delivery of antisense oligonucleotides by gold nanoparticles. Bioconjug Chem 2008; 19: 1009–1016.

23.

Iwashita

Hiramatsu

Otani

. Polyamidoamine dendron-bearing lipid assemblies: Their morphologies and gene transfection ability. J Biomater Appl 2012; 27: 445–456.

24.

Lvov

Shchukin

Möhwald

. Halloysite clay nanotubes for controlled release of protective agents. ACS Nano 2008; 2: 814–820.

25.

Matsushita-Ishiodori

Kuwabara

Sakakoshi

. Photosensitizing carrier proteins for photoinducible RNA interference. Bioconjug Chem 2011; 22: 2222–2226.

26.

Medarova

Pham

Farrar

. In vivo imaging of siRNA delivery and silencing in tumors. Nat Med 2007; 13: 372–377.

27.

Mesri

Wall

. Cancer gene therapy using a survivin mutant adenovirus. J Clin Invest 2001; 108: 981–990.

28.

Pecot

Calin

Coleman

. RNA interference in the clinic: challenges and future directions. Nat Rev Cancer 2011; 11: 59–67.

29.

Ryan

O’Donovan

Duffy

. Survivin: A new target for anti-cancer therapy. Cancer Treat Rev 2009; 35: 553–562.

30.

Schimmer

. Inhibitor of apoptosis proteins: Translating basic knowledge into clinical practice. Cancer Res 2004; 64: 7183–7190.

31.

Stahel

Zangemeister-Wittke

. Antisense oligonucleotides for cancer therapy—an overview. Lung Cancer 2003; 41 Suppl 1: S81–S88.

32.

Sun

Nettesheim

Liu

. Solution structure of human survivin and its binding interface with Smac/Diablo. Biochemistry 2005; 44: 11–17.

33.

Tamm

Wang

Sausville

. IAP-Family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res 1998; 58: 5315–5320.

34.

Tian

Yin

. Supramolecular assembly and antitumor activity of multiwalled carbon nanotube–camptothecin complexes. J Nanosci Nanotechnol 2011; 11: 953–958.

35.

Trabulo

Cardoso

Santos-Ferreira

. Survivin silencing as a promising strategy to enhance the sensitivity of cancer cells to chemotherapeutic agents. Mol Pharm 2011; 8: 1120–1131.

36.

Vergaro

Abdullayev

Lvov

. Cytocompatibility and uptake of halloysite clay nanotubes. Biomacromolecules 2010; 11: 820–826.

37.

Zeng

. Inorganic nanoparticles as carriers for efficient cellular delivery. Chem Eng Sci 2006; 3: 1027–1040.

38.

Yezhelyev

O’Regan

. Proton-sponge coated quantum dots for siRNA delivery and intracellular imaging. J Am Chem Soc 2008; 130: 9006–9012.