Skip to main content
SpringerLink
Log in

Highly efficient gene silencing and bioimaging based on fluorescent carbon dots in vitro and in vivo

  • Research Article
  • Published:
Nano Research Aims and scope Submit manuscript

Abstract

Small interfering RNA (siRNA) is an attractive therapeutic candidate for sequence-specific gene silencing to treat incurable diseases using small molecule drugs. However, its efficient intracellular delivery has remained a challenge. Here, we have developed a highly biocompatible fluorescent carbon dot (CD), and demonstrate a functional siRNA delivery system that induces efficient gene knockdown in vitro and in vivo. We found that CD nanoparticles (NPs) enhance the cellular uptake of siRNA, via endocytosis in tumor cells, with low cytotoxicity and unexpected immune responses. Real-time study of fluorescence imaging in live cells shows that CD NPs favorably localize in cytoplasm and successfully release siRNA within 12 h. Moreover, we demonstrate that CD NP-mediated siRNA delivery significantly silences green fluorescence protein (GFP) expression and inhibits tumor growth in a breast cancer cell xenograft mouse model of tumor-specific therapy. We have developed a multifunctional siRNA delivery vehicle enabling simultaneous bioimaging and efficient downregulation of gene expression, that shows excellent potential for gene therapy.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Hamilton, A. J.; Baulcombe, D. C. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 1999, 286, 950–952.

    Article  Google Scholar 

  2. Elbashir, S. M.; Harborth, J.; Lendeckel, W.; Yalcin, A.; Weber, K.; Tuschl, T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001, 411, 494–498.

    Article  Google Scholar 

  3. Fire, A.; Xu, S. Q.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Mello, C. C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998, 391, 806–811.

    Article  Google Scholar 

  4. Nykänen, A.; Haley, B.; Zamore, P. D. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 2001, 107, 309–321.

    Article  Google Scholar 

  5. Martinez, J.; Patkaniowska, A.; Urlaub, H.; Lührmann, R.; Tuschl, T. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 2002, 110, 563–574.

    Article  Google Scholar 

  6. Dorsett, T.; Tuschl, T. siRNAs: Applications in functional genomics and potential as therapeutics. Nat. Rev. Drug Discov. 2004, 3, 318–329.

    Article  Google Scholar 

  7. Djiane, A.; Yogev, S.; Mlodzik, M. The apical determinants aPKC and dPatj regulate frizzled-dependent planar cell polarity in the Drosophila eye. Cell 2005, 121, 621–631.

    Article  Google Scholar 

  8. Bumcrot, D.; Manoharan, M.; Koteliansky, V.; Sah, D. W. RNAi therapeutics: A potential new class of pharmaceutical drugs. Nat. Chem. Biol. 2006, 2, 711–719.

    Article  Google Scholar 

  9. Soutschek, J.; Akinc, A.; Bramlage, B.; Charisse, K.; Constien, R.; Donoghue, M.; Elbashir, S.; Geick, A.; Hadwiger, P.; Harborth, J. et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 2004, 432, 173–178.

    Article  Google Scholar 

  10. Dykxhoorn, D. M.; Palliser, D.; Lieberman, J. The silent treatment: siRNAs as small molecule drugs. Gene Ther. 2006, 13, 541–552.

    Article  Google Scholar 

  11. Dykxhoorn, D. M.; Lieberman, J. Running interference: Prospects and obstacles to using small interfering RNAs as small molecule drugs. Annu. Rev. Biomed. Eng. 2006, 8, 377–402.

    Article  Google Scholar 

  12. Whitehead, K. A.; Langer, R.; Anderson, D. G. Knocking down barriers: Advances in siRNA delivery. Nat. Rev. Drug Discov. 2009, 8, 129–138.

    Article  Google Scholar 

  13. Niikura, K.; Kobayashi, K.; Takeuchi, C.; Fujitani, N.; Takahara, S.; Ninomiya, T.; Hagiwara, K.; Mitomo, H.; Ito, Y.; Osada, Y. et al. Amphiphilic gold nanoparticles displaying flexible bifurcated ligands as a carrier for siRNA delivery into the cell cytosol. ACS Appl. Mater. Interfaces 2014, 6, 22146–22154.

    Article  Google Scholar 

  14. Zheng, D.; Giljohann, D. A.; Chen, D. L.; Massich, M. D.; Wang, X. Q.; Iordanov, H.; Mirkin, C. A.; Paller, A. S. Topical delivery of siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation. Proc. Natl. Acad. Sci. USA 2012, 109, 11975–11980.

    Article  Google Scholar 

  15. Lee, J. H.; Lee, K.; Moon, S. H.; Lee, Y.; Park, T. G.; Cheon, J. All-in-one target-cell-specific magnetic nanoparticles for simultaneous molecular imaging and siRNA delivery. Angew. Chem., Int. Ed. 2009, 48, 4174–4179.

    Article  Google Scholar 

  16. Derfus, A. M.; Chen, A. A.; Min, D. H.; Ruoslahti, E.; Bhatia, S. N. Targeted quantum dot conjugates for siRNA delivery. Bioconjugate Chem. 2007, 18, 1391–1396.

    Article  Google Scholar 

  17. Lee, H.; Kim, I. K.; Park, T. G. Intracellular trafficking and unpacking of siRNA/quantum dot-PEI complexes modified with and without cell penetrating peptide: Confocal and flow cytometric FRET analysis. Bioconjugate Chem. 2010, 21, 289–295.

    Article  Google Scholar 

  18. Na, H. K.; Kim, M. H.; Park, K.; Ryoo, S. R.; Lee, K. E.; Jeon, H.; Ryoo, R.; Hyeon, C.; Min, D. H. Efficient functional delivery of siRNA using mesoporous silica nanoparticles with ultralarge pores. Small 2012, 8, 1752–1761.

    Article  Google Scholar 

  19. Urban-Klein, B.; Werth, S.; Abuharbeid, S.; Czubayko, F.; Aigner, A. RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo. Gene Ther. 2005, 12, 461–466.

    Article  Google Scholar 

  20. Yano, J.; Hirabayashi, K.; Nakagawa, S.; Yamaguchi, T.; Nogawa, M.; Kashimori, I.; Naito, H.; Kitagawa, H.; Ishiyama, K.; Ohgi, T. et al. Antitumor activity of small interfering RNA/cationic liposome complex in mouse models of cancer. Clin. Cancer Res. 2004, 10, 7721–7726.

    Article  Google Scholar 

  21. Sun, C. Y.; Shen, S.; Xu, C. F.; Li, H. J.; Liu, Y.; Cao, Z. T.; Yang, X. Z.; Xia, J. X.; Wang, J. Tumor acidity-sensitive polymeric vector for active targeted siRNA delivery. J. Am. Soc. Chem. 2015, 137, 15217–15224.

    Article  Google Scholar 

  22. Ngamcherdtrakul, W.; Morry, J.; Gu, S. D.; Castro, D. J.; Goodyear, S. M.; Sangvanich, T.; Reda, M. M.; Lee, R.; Mihelic, S. A.; Beckman, B. L. et al. Cationic polymer modified mesoporous Silica nanoparticles for targeted siRNA delivery to HER2+ breast cancer. Adv. Funct. Mater. 2015, 25, 2646–2659.

    Article  Google Scholar 

  23. Lv, H. T.; Zhang, S. B.; Wang, B.; Cui, S. H.; Yan, J. Toxicity of cationic lipids and cationic polymers in gene delivery. J. Control. Release 2006, 114, 100–109.

    Article  Google Scholar 

  24. Yang, S. T.; Cao, L.; Luo, P. G.; Lu, F. S.; Wang, X.; Wang, H. F.; Meziani, M. J.; Liu, Y. F.; Qi, G.; Sun, Y. P. Carbon dots for optical imaging in vivo. J. Am. Chem. Soc. 2009, 131, 11308–11309.

    Article  Google Scholar 

  25. Baker, S. N.; Baker, G. A. Luminescent carbon nanodots: Emergent nanolights. Angew. Chem., Int. Ed. 2010, 49, 6726–6744.

    Article  Google Scholar 

  26. Liu, C. J.; Zhang, P.; Tian, F.; Li, W. C.; Li, F.; Liu, W. G. One-step synthesis of surface passivated carbon nanodots by microwave assisted pyrolysis for enhanced multicolor photoluminescence and bioimaging. J. Mater. Chem. 2011, 21, 13163–13167.

    Article  Google Scholar 

  27. Li, H. T.; Kang, Z. H.; Liu, Y.; Lee, S. T. Carbon nanodots: Synthesis, properties and applications. J. Mater. Chem. 2012, 22, 24230–24253.

    Article  Google Scholar 

  28. Zhai, X. Y.; Zhang, P.; Liu, C. J.; Bai, T.; Li, W. C.; Dai, L. M.; Liu, W. G. Highly luminescent carbon nanodots by microwave-assisted pyrolysis. Chem. Commun. 2012, 48, 7955–7957.

    Article  Google Scholar 

  29. Miao, P.; Han, K.; Tang, Y. G.; Wang, B. D.; Lin, T.; Cheng, W. B. Recent advances in carbon nanodots: Synthesis, properties and biomedical applications. Nanoscale 2015, 7, 1586–1595.

    Article  Google Scholar 

  30. Zheng, X. T.; Ananthanarayanan, A.; Luo, K. Q.; Chen, P. Glowing graphene quantum dots and carbon dots: Properties, syntheses, and biological applications. Small 2015, 11, 1620–1636.

    Article  Google Scholar 

  31. Zhang, T. Q.; Liu, X. Y.; Fan, Y.; Guo, X. Y.; Zhou, L.; Lv, Y.; Lin, J. One-step microwave synthesis of N-doped hydroxyl-functionalized carbon dots with ultra-high fluorescence quantum yields. Nanoscale 2016, 8, 15281–15287.

    Article  Google Scholar 

  32. Tang, J.; Kong, B.; Wu, H.; Xu, M.; Wang, Y. C.; Wang, Y. L.; Zhao, D. Y.; Zheng, G. F. Carbon nanodots featuring efficient FRET for real-time monitoring of drug delivery and two-photon imaging. Adv. Mater. 2013, 25, 6569–6574.

    Article  Google Scholar 

  33. Liu, C. J.; Zhang, P.; Zhai, X. Y.; Tian, F.; Li, W. C.; Yang, J. H.; Liu, Y.; Wang, H. B.; Wang, W.; Liu, W. G. Nanocarrier for gene delivery and bioimaging based on carbon dots with PEI-passivation enhanced fluorescence. Biomaterials 2012, 33, 3604–3613.

    Article  Google Scholar 

  34. Hu, L. M.; Sun, Y.; Li, S. L.; Wang, X. L.; Hu, K. L.; Wang, L. R.; Liang, X. J.; Wu, Y. Multifunctional carbon dots with high quantum yield for imaging and gene delivery. Carbon 2014, 67, 508–513.

    Article  Google Scholar 

  35. Cao, L.; Wang, X.; Meziani, M. J.; Lu, F. S.; Wang, H. F.; Luo, P. G.; Lin, Y.; Harruff, B. A.; Veca, L. M.; Murray, D. et al. Carbon dots for multiphoton bioimaging. J. Am. Chem. Soc. 2007, 129, 11318–11319.

    Article  Google Scholar 

  36. Zhu, S. J.; Meng, Q. N.; Wang, L.; Zhang, J. H.; Song, Y. B.; Jin, H.; Zhang, K.; Sun, H. C.; Wang, H. Y.; Yang, B. Highly photoluminescent carbon dots for multicolor patterning, sensors, and bioimaging. Angew. Chem., Int. Ed. 2013, 52, 3953–3957.

    Article  Google Scholar 

  37. Zhu, A. W.; Qu, Q.; Shao, X. L.; Kong, B.; Tian, Y. Carbon-dot-based dual-emission nanohybrid produces a ratiometric fluorescent sensor for in vivo imaging of cellular copper ions. Angew. Chem., Int. Ed. 2012, 51, 7185–7189.

    Article  Google Scholar 

  38. Huang, P.; Lin, J.; Wang, X. S.; Wang, Z.; Zhang, C. L.; He, M.; Wang, K.; Chen, F.; Li, Z. M.; Shen, G. X. et al. Light-triggered theranostics based on photosensitizerconjugated carbon dots for simultaneous enhancedfluorescence imaging and photodynamic therapy. Adv. Mater. 2012, 24, 5104–5110.

    Article  Google Scholar 

  39. Hola, K.; Zhang, Y.; Wang, Y.; Giannelis, E. P.; Zboril, R.; Rogach, A. L. Carbon dots—Emerging light emitters for bioimaging, cancer therapy and optoelectronics. Nano Today 2014, 9, 590–603.

    Article  Google Scholar 

  40. Chen, D. Q.; Dougherty, C. A.; Zhu, K. C.; Hong, H. Theranostic applications of carbon nanomaterials in cancer: Focus on imaging and cargo delivery. J. Control. Release 2015, 210, 230–245.

    Article  Google Scholar 

  41. Dong, Y. Q.; Wang, R. X.; Li, H.; Shao, J. W.; Chi, Y. W.; Lin, X. M.; Chen, G. N. Polyamine-functionalized carbon quantum dots for chemical sensing. Carbon 2012, 50, 2810–2815.

    Article  Google Scholar 

  42. Yu, P.; Wen, X. M.; Toh, Y. R.; Tang, J. Temperaturedependent fluorescence in carbon dots. J. Phys. Chem. C 2012, 116, 25552–25557.

    Article  Google Scholar 

  43. Mei, Q. S.; Zhang, K.; Guan, G. J.; Liu, B. H.; Wang, S. H.; Zhang, Z. P. Highly efficient photoluminescent graphene oxide with tunable surface properties. Chem. Commun. 2010, 46, 7319–7321.

    Article  Google Scholar 

  44. Kim, H.; Kim, W. J. Photothermally controlled gene delivery by reduced graphene oxide-polyethylenimine nanocomposite. Small 2014, 10, 117–126.

    Article  Google Scholar 

  45. Dong, Y. Q.; Pang, H. C.; Yang, H. B.; Guo, C. X.; Shao, J. W.; Chi, Y. W.; Li, C. M.; Yu, T. Carbon-based dots co-doped with nitrogen and sulfur for high quantum yield and excitation-independent emission. Angew. Chem., Int. Ed. 2013, 52, 7800–7804.

    Article  Google Scholar 

  46. Singha, K.; Namgung, R.; Kim, W. J. Polymers in smallinterfering RNA delivery. Nucleic Acid Ther. 2011, 21, 133–147.

    Article  Google Scholar 

  47. Bieber, T.; Elsä sser, H. P. Preparation of a low molecular weight polyethylenimine for efficient cell transfection. Biotechniques 2001, 30, 74–77, 80–81.

    Google Scholar 

  48. Gosselin, M. A.; Guo, W. J.; Lee, R. J. Efficient gene transfer using reversibly cross-linked low molecular weight polyethylenimine. Bioconjugate Chem. 2001, 12, 989–994.

    Article  Google Scholar 

  49. Hu, C.; Peng, Q.; Chen, F. J.; Zhong, Z. L.; Zhuo, R. X. Low molecular weight polyethylenimine conjugated gold nanoparticles as efficient gene vectors. Bioconjugate Chem. 2010, 21, 836–843.

    Article  Google Scholar 

  50. Nunes, A.; Amsharov, N.; Guo, C.; Van den Bossche, J.; Santhosh, P.; Karachalios, T. K.; Nitodas, S. F.; Burghard, M.; Kostarelos, K.; Al-Jamal, K. T. Hybrid polymer-grafted multiwalled carbon nanotubes for in vitro gene delivery. Small 2010, 6, 2281–2291.

    Article  Google Scholar 

  51. Boussif, O.; Lezoualc’h, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. Proc. Natl. Acad. Sci. USA 1995, 92, 7297–7301.

    Article  Google Scholar 

  52. Fischer, D.; Bieber, T.; Li, Y. X.; Elsässer, H. P.; Kissel, T. A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: Effect of molecular weight on transfection efficiency and cytotoxicity. Pharm. Res. 1999, 16, 1273–1279.

    Article  Google Scholar 

  53. Sharma, V. K.; Thomas, M.; Klibanov, A. M. Mechanistic studies on aggregation of polyethylenimine-DNA complexes and its prevention. Biotechnol. Bioeng. 2005, 90, 614–620.

    Article  Google Scholar 

  54. Wang, X. L.; Zhou, L. Z.; Ma, Y. J.; Li, X.; Gu, H. C. Control of aggregate size of polyethyleneimine-coated magnetic nanoparticles for magnetofection. Nano Res. 2009, 2, 365–372.

    Article  Google Scholar 

  55. Chen, H. H.; Ho, Y. P.; Jiang, X.; Mao, H. Q.; Wang, T. H.; Leong, K. W. Quantitative comparison of intracellular unpacking kinetics of polyplexes by a model constructed from quantum dot-FRET. Mol. Ther. 2008, 16, 324–332.

    Article  Google Scholar 

  56. Ferrara, N. The role of vascular endothelial growth factor in pathological angiogenesis. Breast Cancer Res. Treat. 1995, 36, 127–137.

    Article  Google Scholar 

  57. Takei, Y.; Kadomatsu, K.; Yuzawa, Y.; Matsuo, S.; Muramatsu, T. A small interfering RNA targeting vascular endothelial growth factor as cancer therapeutics. Cancer Res. 2004, 64, 3365–3370.

    Article  Google Scholar 

  58. Alexopoulou, L.; Holt, A. C.; Medzhitov, R.; Flavell, R. A. Recognition of double-stranded RNA and activation of NF-B by Toll-like receptor 3. Nature 2001, 413, 732–738.

    Article  Google Scholar 

  59. Matsumoto, M.; Seya, T. TLR3: Interferon induction by double-stranded RNA including poly(I: C). Adv. Drug Deliv. Rev. 2008, 60, 805–812.

    Article  Google Scholar 

  60. Ryoo, S. R.; Jang, H.; Kim, K. S.; Lee, B.; Kim, K. B.; Kim, Y. K.; Yeo, W. S.; Lee, Y.; Kim, D. E.; Min, D. H. Functional delivery of DNAzyme with iron oxide nanoparticles for hepatitis C virus gene knockdown. Biomaterials 2012, 33, 2754–2761.

    Article  Google Scholar 

  61. Lammers, T.; Peschke, P.; Kühnlein, R.; Subr, V.; Ulbrich, K.; Huber, P.; Hennink, W.; Storm, G. Effect of intratumoral injection on the biodistribution, the therapeutic potential of HPMA copolymer-based drug delivery systems. Neoplasia 2006, 8, 788–795.

    Article  Google Scholar 

  62. Moon, H. K.; Lee, S. H.; Choi, H. C. In vivo near-infrared mediated tumor destruction by photothermal effect of carbon nanotubes. ACS Nano 2009, 3, 3707–3713.

    Article  Google Scholar 

  63. Almeida, J. P. M.; Chen, A. L.; Foster, A.; Drezek, R. In vivo biodistribution of nanoparticles. Nanomedicine 2011, 6, 815–835.

    Article  Google Scholar 

  64. Petros, R. A.; DeSimone, J. M. Strategies in the design of nanoparticles for therapeutic applications. Nat. Rev. Drug Discov. 2010, 9, 615–627.

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the Basic Science Research Program (Nos. 2011-0017356 and 2011-0020322), International S&T Cooperation Program (No. 2014K1B1A1073716) and the Research Center Program (No. IBS-R008-D1) of IBS (Institute for Basic Science) through the National Research Foundation of Korea (NRF) funded by the Korean government (MEST).

Author information

Authors and Affiliations

  1. Center for RNA Research, Institute for Basic Sciences (IBS), Department of Chemistry, Seoul National University, Seoul, 08826, Republic of Korea

    Seongchan Kim & Dal-Hee Min

  2. Department of Energy Engineering and Department of Chemistry, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 44919, Republic of Korea

    Yuri Choi & Byeong-Su Kim

  3. Institute of Nanobio Convergence Technology, Lemonex Inc., Seoul, 08826, Republic of Korea

    Ginam Park, Cheolhee Won & Dal-Hee Min

  4. College of Pharmacy, Ajou University, Worldcuplo 206, Yeongtong-gu, Suwon, 16499, Republic of Korea

    Young-Joon Park

  5. Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, Republic of Korea

    Younghoon Lee

Authors
  1. Seongchan Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yuri Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ginam Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Cheolhee Won
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Young-Joon Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Younghoon Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Byeong-Su Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Dal-Hee Min
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Younghoon Lee, Byeong-Su Kim or Dal-Hee Min.

Additional information

These authors contributed equally to this work.

Highly efficient gene silencing and bioimaging based on fluorescent carbon dots in vitro and in vivo

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kim, S., Choi, Y., Park, G. et al. Highly efficient gene silencing and bioimaging based on fluorescent carbon dots in vitro and in vivo . Nano Res. 10, 503–519 (2017). https://doi.org/10.1007/s12274-016-1309-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12274-016-1309-1

Keywords

Search

Navigation