BACKGROUND: Suitable fluorophores are the core of fluorescence imaging. Among the most exciting, yet controversial, labels are quantum dots (QDs) with their unique optical and chemical properties, but also considerable toxicity. This hinders QDs applicability in living systems. Surface chemistry has a profound impact on biological behavior of QDs. This study describes a two-step synthesis of QDs formed by CdTe core doped with Schiff base ligand for lanthanides [Ln (Yb3+, Tb3+ and Gd3+)] as novel cytocompatible fluorophores. RESULTS: Microwave-assisted synthesis resulted in water-soluble nanocrystals with high colloidal and fluorescence stability with quantum yields of 40.9-58.0%. Despite induction of endocytosis and cytoplasm accumulation of Yb- and TbQDs, surface doping resulted in significant enhancement in cytocompatibility when compared to the un-doped CdTe QDs. Furthermore, only negligible antimigratory properties without triggering formation of reactive oxygen species were found, particularly for TbQDs. Ln-doped QDs did not cause observable hemolysis, adsorbed only a low degree of plasma proteins onto their surface and did not possess significant genotoxicity. To validate the applicability of Ln-doped QDs for in vitro visualization of receptor status of living cells, we performed a site-directed conjugation of antibodies towards immuno-labeling of clinically relevant target-human norepinephrine transporter (hNET), over-expressed in neuroendocrine tumors like neuroblastoma. Immuno-performance of modified TbQDs was successfully tested in distinct types of cells varying in hNET expression and also in neuroblastoma cells with hNET expression up-regulated by vorinostat. CONCLUSION: For the first time we show that Ln-doping of CdTe QDs can significantly alleviate their cytotoxic effects. The obtained results imply great potential of Ln-doped QDs as cytocompatible and stable fluorophores for various bio-labeling applications.

Central European Institute of Technology Brno University of Technology Purkynova 123 612 00 Brno Czech Republic

Department of Chemistry and Biochemistry Mendel University in Brno Zemedelska 1 613 00 Brno Czech Republic

Materials Research Centre Faculty of Chemistry Brno University of Technology Purkynova 118 612 00 Brno Czech Republic

Zobrazit více v PubMed

Hardman R. A toxicologic review of quantum dots: toxicity depends on physicochemical and environmental factors. Environ Health Perspect. 2006;114:165–172. doi: 10.1289/ehp.8284. PubMed DOI PMC

Wu XY, Liu HJ, Liu JQ, Haley KN, Treadway JA, Larson JP, Ge NF, Peale F, Bruchez MP. Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat Biotechnol. 2003;21:452. doi: 10.1038/nbt0403-452b. PubMed DOI

Gao X, Yang L, Petros JA, Marshall FF, Simons JW, Nie S. In vivo molecular and cellular imaging with quantum dots. Curr Opin Biotechnol. 2005;16:63–72. doi: 10.1016/j.copbio.2004.11.003. PubMed DOI

Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, Sundaresan G, Wu AM, Gambhir SS, Weiss S. Quantum dots for live cells, in vivo imaging, and diagnostics. Science. 2005;307:538–544. doi: 10.1126/science.1104274. PubMed DOI PMC

Zrazhevskiy P, Sena M, Gao XH. Designing multifunctional quantum dots for bioimaging, detection, and drug delivery. Chem Soc Rev. 2010;39:4326–4354. doi: 10.1039/b915139g. PubMed DOI PMC

Kovalenko MV, Bodnarchuk MI, Zaumseil J, Lee JS, Talapin DV. Expanding the chemical versatility of colloidal nanocrystals capped with molecular metal chalcogenide ligands. J Am Chem Soc. 2010;132:10085–10092. doi: 10.1021/ja1024832. PubMed DOI

Lovrić J, Bazzi HS, Cuie Y, Fortin GRA, Winnik FM, Maysinger D. Differences in subcellular distribution and toxicity of green and red emitting CdTe quantum dots. J Mol Med. 2005;83:377–385. doi: 10.1007/s00109-004-0629-x. PubMed DOI

Bradburne CE, Delehanty JB, Gemmill KB, Mei BC, Mattoussi H, Susumu K, Blanco-Canosa JB, Dawson PE, Medintz IL. Cytotoxicity of quantum dots used for in vitro cellular labeling: role of QD surface ligand, delivery modality, cell type, and direct comparison to organic fluorophores. Bioconjug Chem. 2013;24:1570–1583. doi: 10.1021/bc4001917. PubMed DOI

Yong KT, Law WC, Roy I, Ling Z, Huang HJ, Swihart MT, Prasad PN. Aqueous phase synthesis of CdTe quantum dots for biophotonics. J Biophotonics. 2011;4:9–20. doi: 10.1002/jbio.201000080. PubMed DOI

Taniguchi S, Green M. The synthesis of CdTe/ZnS core/shell quantum dots using molecular single-source precursors. J Mater Chem C. 2015;3:8425–8433. doi: 10.1039/C5TC01808K. DOI

Lovric J, Cho SJ, Winnik FM, Maysinger D. Unmodified cadmium telluride quantum dots induce reactive oxygen species formation leading to multiple organelle damage and cell death. Chem Biol. 2005;12:1227–1234. doi: 10.1016/j.chembiol.2005.09.008. PubMed DOI

Cho SJ, Maysinger D, Jain M, Roder B, Hackbarth S, Winnik FM. Long-term exposure to CdTe quantum dots causes functional impairments in live cells. Langmuir. 2007;23:1974–1980. doi: 10.1021/la060093j. PubMed DOI

Baker SN, Baker GA. Luminescent carbon nanodots: emergent nanolights. Angew Chem-Int Edit. 2010;49:6726–6744. doi: 10.1002/anie.200906623. PubMed DOI

Hoshino A, Hanada S, Yamamoto K. Toxicity of nanocrystal quantum dots: the relevance of surface modifications. Arch Toxicol. 2011;85:707–720. doi: 10.1007/s00204-011-0695-0. PubMed DOI

Reisfeld R, Gaft M, Saridarov T, Panczer G, Zelner M. Nanoparticles of cadmium sulfide with europium and terbium in zirconia films having intensified luminescence. Mater Lett. 2000;45:154–156. doi: 10.1016/S0167-577X(00)00096-3. DOI

Tiseanu C, Mehra RK, Kho R, Kumke M. Optical properties of terbium-doped thiosalicylic-capped CdS nanocrystals. Chem Phys Lett. 2003;377:131–136. doi: 10.1016/S0009-2614(03)01119-9. DOI

Wang YB, Liang XH, Liu EZ, Hu XY, Fan J. Incorporation of lanthanide (Eu3+) ions in ZnS semiconductor quantum dots with a trapped-dopant model and their photoluminescence spectroscopy study. Nanotechnology. 2015;26:8. PubMed

Cheng L, Yang K, Shao MW, Lu XH, Liu Z. In vivo pharmacokinetics, long-term biodistribution and toxicology study of functionalized upconversion nanoparticles in mice. Nanomedicine. 2011;6:1327–1340. doi: 10.2217/nnm.11.56. PubMed DOI

Misiak M, Skowicki M, Lipinski T, Kowalczyk A, Prorok K, Arabasz S, Bednarkiewicz A. Biofunctionalized upconverting CaF2:Yb, Tm nanoparticles for Candida albicans detection and imaging. Nano Res. 2017;10:3333–3345. doi: 10.1007/s12274-017-1546-y. DOI

Portioli C, Pedroni M, Benati D, Donini M, Bonafede R, Mariotti R, Perbellini L, Cerpelloni M, Dusi S, Speghini A, Bentivoglio M. Citrate-stabilized lanthanide-doped nanoparticles: brain penetration and interaction with immune cells and neurons. Nanomedicine. 2016;11:13. doi: 10.2217/nnm-2016-0297. PubMed DOI

Zhang H, Huang R, Cheung NKV, Guo H, Zanzonico PB, Thaler HT, Lewis JS, Blasberg RG. Imaging the norepinephrine transporter in neuroblastoma: a comparison of [18F]-MFBG and 123I-MIBG. Clin Cancer Res. 2014;20:2182–2191. doi: 10.1158/1078-0432.CCR-13-1153. PubMed DOI PMC

Xiao Y, Forry SP, Gao X, Holbrook RD, Telford WG, Tona A. Dynamics and mechanisms of quantum dot nanoparticle cellular uptake. J Nanobiotechnol. 2010;8:13. doi: 10.1186/1477-3155-8-13. PubMed DOI PMC

Zhang LW, Monteiro-Riviere NA. Mechanisms of quantum dot nanoparticle cellular uptake. Toxicol Sci. 2009;110:138–155. doi: 10.1093/toxsci/kfp087. PubMed DOI

van Nes J, Chan A, van Groningen T, van Sluis P, Koster J, Versteeg R. A NOTCH3 transcriptional module induces cell motility in neuroblastoma. Clin Cancer Res. 2013;19:3485–3494. doi: 10.1158/1078-0432.CCR-12-3021. PubMed DOI

Rzigalinski BA, Strobl JS. Cadmium-containing nanoparticles: perspectives on pharmacology and toxicology of quantum dots. Toxicol Appl Pharmacol. 2009;238:280–288. doi: 10.1016/j.taap.2009.04.010. PubMed DOI PMC

Yan M, Zhang Y, Qin HY, Liu KZ, Guo M, Ge YK, Xu MG, Sun YH, Zheng XX. Cytotoxicity of CdTe quantum dots in human umbilical vein endothelial cells: the involvement of cellular uptake and induction of pro-apoptotic endoplasmic reticulum stress. Int J Nanomed. 2016;11:529–542. PubMed PMC

Oh E, Liu R, Nel A, Gemill KB, Bilal M, Cohen Y, Medintz IL. Meta-analysis of cellular toxicity for cadmium-containing quantum dots. Nat Nanotechnol. 2016;11:479–486. doi: 10.1038/nnano.2015.338. PubMed DOI

Qiu JC, Zhang RB, Li JH, Sang YH, Tang W, Gil PR, Liu H. Fluorescent graphene quantum dots as traceable, pH-sensitive drug delivery systems. Int J Nanomed. 2015;10:6709–6724. PubMed PMC

Derfus AM, Chan WCW, Bhatia SN. Probing the cytotoxicity of semiconductor quantum dots. Nano Lett. 2004;4:11–18. doi: 10.1021/nl0347334. PubMed DOI PMC

Zhang ZY, Berg A, Levanon H, Fessenden RW, Meisel D. On the interactions of free radicals with gold nanoparticles. J Am Chem Soc. 2003;125:7959–7963. doi: 10.1021/ja034830z. PubMed DOI

Lai L, Lin C, Xu ZQ, Han XL, Tian FF, Mei P, Li DW, Ge YS, Jiang FL, Zhang YZ, Liu Y. Spectroscopic studies on the interactions between CdTe quantum dots coated with different ligands and human serum albumin. Spectroc Acta Pt A-Molec Biomol Spectr. 2012;97:366–376. doi: 10.1016/j.saa.2012.06.025. PubMed DOI

Xia Q, Feng XD, Huang HF, Du LY, Yang XD, Wang K. Gadolinium-induced oxidative stress triggers endoplasmic reticulum stress in rat cortical neurons. J Neurochem. 2011;117:38–47. doi: 10.1111/j.1471-4159.2010.07162.x. PubMed DOI

Borm PJA, Muller-Schulte D. Nanoparticles in drug delivery and environmental exposure: same size, same risks? Nanomedicine. 2006;1:235–249. doi: 10.2217/17435889.1.2.235. PubMed DOI

Jan KM, Chien S. Role of surface electric charge in red blood–cell intractions. J Gen Physiol. 1973;61:638–654. doi: 10.1085/jgp.61.5.638. PubMed DOI PMC

Fernandez EL, Gustafson AL, Andersson M, Hellman B, Dencker L. Cadmium-induced changes in apoptotic gene expression levels and DNA damage in mouse embryos are blocked by zinc. Toxicol Sci. 2003;76:162–170. doi: 10.1093/toxsci/kfg208. PubMed DOI

Tang S, Cai QS, Chibli H, Allagadda V, Nadeau JL, Mayer GD. Cadmium sulfate and CdTe-quantum dots alter DNA repair in zebrafish (Danio rerio) liver cells. Toxicol Appl Pharmacol. 2013;272:443–452. doi: 10.1016/j.taap.2013.06.004. PubMed DOI

Ritz S, Schottler S, Kotman N, Baier G, Musyanovych A, Kuharev J, Landfester K, Schild H, Jahn O, Tenzer S, Mailander V. Protein corona of nanoparticles: distinct proteins regulate the cellular uptake. Biomacromolecules. 2015;16:1311–1321. doi: 10.1021/acs.biomac.5b00108. PubMed DOI

Li SC, Wang Y, Wang HT, Bai YF, Liang GF, Wang YY, Huang NP, Xiao ZD. MicroRNAs as participants in cytotoxicity of CdTe quantum dots in NIH/3T3 cells. Biomaterials. 2011;32:3807–3814. doi: 10.1016/j.biomaterials.2011.01.074. PubMed DOI

Tian X, Xiao BB, Wu AQ, Yu L, Zhou JD, Wang Y, Wang N, Guan H, Shang ZF. Hydroxylated-graphene quantum dots induce cells senescence in both p53-dependent and -independent manner. Toxicol Res. 2016;5:1639–1648. doi: 10.1039/C6TX00209A. PubMed DOI PMC

Choi AO, Brown SE, Szyf M, Maysinger D. Quantum dot-induced epigenetic and genotoxic changes in human breast cancer cells. J Mol Med. 2008;86:291–302. doi: 10.1007/s00109-007-0274-2. PubMed DOI

Dostalova S, Cerna T, Hynek D, Koudelkova Z, Vaculovic T, Kopel P, Hrabeta J, Heger Z, Vaculovicova M, Eckschlager T, et al. Site-directed conjugation of antibodies to apoferritin nanocarrier for targeted drug delivery to prostate cancer cells. ACS Appl Mater Interfaces. 2016;8:14430–14441. doi: 10.1021/acsami.6b04286. PubMed DOI

Heger Z, Cernei N, Krizkova S, Masarik M, Kopel P, Hodek P, Zitka O, Adam V, Kizek R. Paramagnetic nanoparticles as a platform for FRET-based sarcosine picomolar detection. Sci Rep. 2015;5:1–8. doi: 10.1038/srep08868. PubMed DOI PMC

Streby KA, Shah N, Ranalli MA, Kunkler A, Cripe TP. Nothing but NET: a review of norepinephrine transporter expression and efficacy of I-131-mIBG therapy. Pediatr Blood Cancer. 2015;62:5–11. doi: 10.1002/pbc.25200. PubMed DOI PMC

Ettinger A, Wittmann T. Fluorescence live cell imaging. In: Waters JC, Wittmann T, editors. Quantitative imaging in cell biology. San Diego: Elsevier Academic Press Inc; 2014. pp. 77–94.

Kopel P, Dolezal K, Langer V, Jun D, Adam V, Kuca K, Kizek R. Trithiocyanurate complexes of iron, manganese and nickel and their anticholinesterase activity. Molecules. 2014;19:4338–4354. doi: 10.3390/molecules19044338. PubMed DOI PMC

Moulick A, Blazkova I, Milosavljevic V, Fohlerova Z, Hubalek J, Kopel P, Vaculovicova M, Adam V, Kizek R. Application of CdTe/ZnSe quantum dots in in vitro imaging of chicken tissue and embryo. Photochem Photobiol. 2015;91:417–423. doi: 10.1111/php.12398. PubMed DOI

Pradhan N, Peng XG. Efficient and color-tunable Mn-doped ZnSe nanocrystal emitters: control of optical performance via greener synthetic chemistry. J Am Chem Soc. 2007;129:3339–3347. doi: 10.1021/ja068360v. PubMed DOI

Evans BC, Nelson CE, Yu SS, Beavers KR, Kim AJ, Li H, Nelson HM, Giorgio TD, Duvall CL. Ex vivo red blood cell hemolysis assay for the evaluation of pH-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs. J Vis Exp. 2013;73:1–5. PubMed PMC

Norepinephrine transporter-derived homing peptides enable rapid endocytosis of drug delivery nanovehicles into neuroblastoma cells