Nano Research

, Volume 10, Issue 2, pp 503–519 | Cite as

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

  • Seongchan Kim
  • Yuri Choi
  • Ginam Park
  • Cheolhee Won
  • Young-Joon Park
  • Younghoon LeeEmail author
  • Byeong-Su KimEmail author
  • Dal-Hee MinEmail author
Research Article


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.


bioimaging carbon dot gene delivery RNA interference targeted cancer therapy 


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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).

Supplementary material

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  1. [1]
    Hamilton, A. J.; Baulcombe, D. C. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 1999, 286, 950–952.CrossRefGoogle Scholar
  2. [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.CrossRefGoogle Scholar
  3. [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.CrossRefGoogle Scholar
  4. [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.CrossRefGoogle Scholar
  5. [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.CrossRefGoogle Scholar
  6. [6]
    Dorsett, T.; Tuschl, T. siRNAs: Applications in functional genomics and potential as therapeutics. Nat. Rev. Drug Discov. 2004, 3, 318–329.CrossRefGoogle Scholar
  7. [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.CrossRefGoogle Scholar
  8. [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.CrossRefGoogle Scholar
  9. [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.CrossRefGoogle Scholar
  10. [10]
    Dykxhoorn, D. M.; Palliser, D.; Lieberman, J. The silent treatment: siRNAs as small molecule drugs. Gene Ther. 2006, 13, 541–552.CrossRefGoogle Scholar
  11. [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.CrossRefGoogle Scholar
  12. [12]
    Whitehead, K. A.; Langer, R.; Anderson, D. G. Knocking down barriers: Advances in siRNA delivery. Nat. Rev. Drug Discov. 2009, 8, 129–138.CrossRefGoogle Scholar
  13. [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.CrossRefGoogle Scholar
  14. [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.CrossRefGoogle Scholar
  15. [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.CrossRefGoogle Scholar
  16. [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.CrossRefGoogle Scholar
  17. [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.CrossRefGoogle Scholar
  18. [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.CrossRefGoogle Scholar
  19. [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.CrossRefGoogle Scholar
  20. [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.CrossRefGoogle Scholar
  21. [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.CrossRefGoogle Scholar
  22. [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.CrossRefGoogle Scholar
  23. [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.CrossRefGoogle Scholar
  24. [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.CrossRefGoogle Scholar
  25. [25]
    Baker, S. N.; Baker, G. A. Luminescent carbon nanodots: Emergent nanolights. Angew. Chem., Int. Ed. 2010, 49, 6726–6744.CrossRefGoogle Scholar
  26. [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.CrossRefGoogle Scholar
  27. [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.CrossRefGoogle Scholar
  28. [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.CrossRefGoogle Scholar
  29. [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.CrossRefGoogle Scholar
  30. [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.CrossRefGoogle Scholar
  31. [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.CrossRefGoogle Scholar
  32. [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.CrossRefGoogle Scholar
  33. [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.CrossRefGoogle Scholar
  34. [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.CrossRefGoogle Scholar
  35. [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.CrossRefGoogle Scholar
  36. [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.CrossRefGoogle Scholar
  37. [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.CrossRefGoogle Scholar
  38. [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.CrossRefGoogle Scholar
  39. [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.CrossRefGoogle Scholar
  40. [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.CrossRefGoogle Scholar
  41. [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.CrossRefGoogle Scholar
  42. [42]
    Yu, P.; Wen, X. M.; Toh, Y. R.; Tang, J. Temperaturedependent fluorescence in carbon dots. J. Phys. Chem. C 2012, 116, 25552–25557.CrossRefGoogle Scholar
  43. [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.CrossRefGoogle Scholar
  44. [44]
    Kim, H.; Kim, W. J. Photothermally controlled gene delivery by reduced graphene oxide-polyethylenimine nanocomposite. Small 2014, 10, 117–126.CrossRefGoogle Scholar
  45. [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.CrossRefGoogle Scholar
  46. [46]
    Singha, K.; Namgung, R.; Kim, W. J. Polymers in smallinterfering RNA delivery. Nucleic Acid Ther. 2011, 21, 133–147.CrossRefGoogle Scholar
  47. [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. [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.CrossRefGoogle Scholar
  49. [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.CrossRefGoogle Scholar
  50. [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.CrossRefGoogle Scholar
  51. [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.CrossRefGoogle Scholar
  52. [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.CrossRefGoogle Scholar
  53. [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.CrossRefGoogle Scholar
  54. [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.CrossRefGoogle Scholar
  55. [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.CrossRefGoogle Scholar
  56. [56]
    Ferrara, N. The role of vascular endothelial growth factor in pathological angiogenesis. Breast Cancer Res. Treat. 1995, 36, 127–137.CrossRefGoogle Scholar
  57. [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.CrossRefGoogle Scholar
  58. [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.CrossRefGoogle Scholar
  59. [59]
    Matsumoto, M.; Seya, T. TLR3: Interferon induction by double-stranded RNA including poly(I: C). Adv. Drug Deliv. Rev. 2008, 60, 805–812.CrossRefGoogle Scholar
  60. [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.CrossRefGoogle Scholar
  61. [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.CrossRefGoogle Scholar
  62. [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.CrossRefGoogle Scholar
  63. [63]
    Almeida, J. P. M.; Chen, A. L.; Foster, A.; Drezek, R. In vivo biodistribution of nanoparticles. Nanomedicine 2011, 6, 815–835.CrossRefGoogle Scholar
  64. [64]
    Petros, R. A.; DeSimone, J. M. Strategies in the design of nanoparticles for therapeutic applications. Nat. Rev. Drug Discov. 2010, 9, 615–627.CrossRefGoogle Scholar

Copyright information

© Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.Center for RNA Research, Institute for Basic Sciences (IBS), Department of ChemistrySeoul National UniversitySeoulRepublic of Korea
  2. 2.Department of Energy Engineering and Department of ChemistryUlsan National Institute of Science and Technology (UNIST)UlsanRepublic of Korea
  3. 3.Institute of Nanobio Convergence TechnologyLemonex Inc.SeoulRepublic of Korea
  4. 4.College of PharmacyAjou UniversitySuwonRepublic of Korea
  5. 5.Department of ChemistryKorea Advanced Institute of Science and Technology (KAIST)DaejeonRepublic of Korea

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