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.
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Hamilton, A. J.; Baulcombe, D. C. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 1999, 286, 950–952.
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.
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.
Nykänen, A.; Haley, B.; Zamore, P. D. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 2001, 107, 309–321.
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.
Dorsett, T.; Tuschl, T. siRNAs: Applications in functional genomics and potential as therapeutics. Nat. Rev. Drug Discov. 2004, 3, 318–329.
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.
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.
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.
Dykxhoorn, D. M.; Palliser, D.; Lieberman, J. The silent treatment: siRNAs as small molecule drugs. Gene Ther. 2006, 13, 541–552.
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.
Whitehead, K. A.; Langer, R.; Anderson, D. G. Knocking down barriers: Advances in siRNA delivery. Nat. Rev. Drug Discov. 2009, 8, 129–138.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Baker, S. N.; Baker, G. A. Luminescent carbon nanodots: Emergent nanolights. Angew. Chem., Int. Ed. 2010, 49, 6726–6744.
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.
Li, H. T.; Kang, Z. H.; Liu, Y.; Lee, S. T. Carbon nanodots: Synthesis, properties and applications. J. Mater. Chem. 2012, 22, 24230–24253.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Yu, P.; Wen, X. M.; Toh, Y. R.; Tang, J. Temperaturedependent fluorescence in carbon dots. J. Phys. Chem. C 2012, 116, 25552–25557.
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.
Kim, H.; Kim, W. J. Photothermally controlled gene delivery by reduced graphene oxide-polyethylenimine nanocomposite. Small 2014, 10, 117–126.
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.
Singha, K.; Namgung, R.; Kim, W. J. Polymers in smallinterfering RNA delivery. Nucleic Acid Ther. 2011, 21, 133–147.
Bieber, T.; Elsä sser, H. P. Preparation of a low molecular weight polyethylenimine for efficient cell transfection. Biotechniques 2001, 30, 74–77, 80–81.
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.
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.
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.
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.
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.
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.
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.
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.
Ferrara, N. The role of vascular endothelial growth factor in pathological angiogenesis. Breast Cancer Res. Treat. 1995, 36, 127–137.
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.
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.
Matsumoto, M.; Seya, T. TLR3: Interferon induction by double-stranded RNA including poly(I: C). Adv. Drug Deliv. Rev. 2008, 60, 805–812.
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.
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.
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.
Almeida, J. P. M.; Chen, A. L.; Foster, A.; Drezek, R. In vivo biodistribution of nanoparticles. Nanomedicine 2011, 6, 815–835.
Petros, R. A.; DeSimone, J. M. Strategies in the design of nanoparticles for therapeutic applications. Nat. Rev. Drug Discov. 2010, 9, 615–627.
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).
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Highly efficient gene silencing and bioimaging based on fluorescent carbon dots in vitro and in vivo
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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
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DOI: https://doi.org/10.1007/s12274-016-1309-1