Cytotoxicity and non-specific cellular uptake of bare and surface-modified upconversion nanoparticles in human skin cells


The cytotoxicity and non-specific cellular uptake of the most popular composition of upconversion nanoparticle (UCNP), NaYF4:Yb3+:Er3+, is reported using normal human skin cells, including dermal fibroblasts and immortalized human epidermal linear keratinocytes (HaCaT). A new hydrophilization reaction of as-synthesized UCNPs based on tetramethylammonium hydroxide (TMAH) enabled evaluation of the intrinsic cytotoxicity of bare UCNPs. The cytotoxicity effects of the UCNP surface-coating and polystyrene host were investigated over the concentration range 62.5–125 μg/mL with 24-h incubation, using a MTT test and optical microscopy. The fibroblast viability was not compromised by UCNPs, whereas the viability of keratinocytes varied from 52% ± 4% to 100% ± 10% than the control group, depending on the surface modification. Bare UCNPs reduced the keratinocyte viability to 76% ± 3%, while exhibiting profound non-specific cellular uptake. Hydrophilic poly(D,L-lactide)- and poly(maleic anhydride-alt-1-octadecene)-coated UCNPs were found to be least cytotoxic among the polymer-coated UCNPs, and were readily internalized by human skin cells. Polystyrene microbeads impregnated with UCNPs remained nontoxic. Surprisingly, no correlation was found between UCNP cytotoxicity and the internalization level in cells, although the latter ranged broadly from 0.03% to 59%, benchmarked against 100% uptake level of TMAH-UCNPs.

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  1. [1]

    Hilderbrand, S. A.; Weissleder, R. Near-infrared fluorescence: Application to in vivo molecular imaging. Curr. Opin. Chem. Biol. 2010, 14, 71–79.

    Article  Google Scholar 

  2. [2]

    Resch-Genger, U.; Grabolle, M.; Cavaliere-Jaricot, S.; Nitschke, R.; Nann, T. Quantum dots versus organic dyes as fluorescent labels. Nat. Methods 2008, 5, 763–775.

    Article  Google Scholar 

  3. [3]

    Yi, G. S.; Lu, H. C.; Zhao, S. Y.; Ge, Y.; Yang, W. J.; Chen, D. P.; Guo, L. H. Synthesis, characterization, and biological application of size-controlled nanocrystalline NaYF4:Yb,Er infrared-to-visible up-conversion phosphors. Nano Lett. 2004, 4, 2191–2196.

    Article  Google Scholar 

  4. [4]

    Edmonds, A. M.; Sobhan, M. A.; Sreenivasan, V. K. A.; Grebenik, E. A.; Rabeau, J. R.; Goldys, E. M.; Zvyagin, A. V. Nano-ruby: A promising fluorescent probe for background-free cellular imaging. Part. Part. Syst. Charact. 2013, 30, 506–513.

    Article  Google Scholar 

  5. [5]

    Lu, Y. Q.; Zhao, J. B.; Zhang, R.; Liu, Y. J.; Liu, D. M.; Goldys, E. M.; Yang, X. S.; Xi, P.; Sunna, A.; Lu, J. et al. Tunable lifetime multiplexing using luminescent nanocrystals. Nat. Photonics 2013, 8, 32–36.

    Article  Google Scholar 

  6. [6]

    Sreenivasan, V. K. A.; Kelf, T. A.; Grebenik, E. A.; Stremovskiy, O. A.; Say, J. M.; Rabeau, J. R.; Zvyagin, A. V.; Deyev, S. M. A modular design of low-background bioassays based on a high-affinity molecular pair barstar: Barnase. Proteomics 2013, 13, 1437–1443.

    Article  Google Scholar 

  7. [7]

    Maeda, H. Tumor-selective delivery of macromolecular drugs via the EPR effect: Background and future prospects. Bioconjugate Chem. 2010, 21, 797–802.

    Article  Google Scholar 

  8. [8]

    Wang, F.; Liu, X. G. Recent advances in the chemistry of lanthanide-doped upconversion nanocrystals. Chem. Soc. Rev. 2009, 38, 976–989.

    Article  Google Scholar 

  9. [9]

    Nyk, M.; Kumar, R.; Ohulchanskyy, T. Y.; Bergey, E. J.; Prasad, P. N. High contrast in vitro and in vivo photoluminescence bioimaging using near infrared to near infrared up-conversion in Tm3+ and Yb3+ doped fluoride nanophosphors. Nano Lett. 2008, 8, 3834–3838.

    Article  Google Scholar 

  10. [10]

    Liu, Q.; Sun, Y.; Yang, T. S.; Feng, W.; Li, C. G.; Li, F. Y. Sub-10 nm hexagonal lanthanide-doped NaLuF4 upconversion nanocrystals for sensitive bioimaging in vivo. J. Am. Chem. Soc. 2011, 133, 17122–17125.

    Article  Google Scholar 

  11. [11]

    Morgan, C. G.; Dad, S.; Mitchell, A. C. Present status of, and future prospects for, upconverting phosphors in proximity-based bioassay. J. Alloys Compd. 2008, 451, 526–529.

    Article  Google Scholar 

  12. [12]

    Wang, C.; Tao, H. Q.; Cheng, L.; Liu, Z. Near-infrared light induced in vivo photodynamic therapy of cancer based on upconversion nanoparticles. Biomaterials 2011, 32, 6145–6154.

    Google Scholar 

  13. [13]

    Min, Y. Z.; Li, J. M.; Liu, F.; Yeow, E. K. L.; Xing, B. G. Near-infrared light-mediated photoactivation of a platinum antitumor prodrug and simultaneous cellular apoptosis imaging by upconversion-luminescent nanoparticles. Angew. Chem. Int. Ed. 2014, 53, 1012–1016.

    Article  Google Scholar 

  14. [14]

    Achatz, D. E.; Meier, R. J.; Fischer, L. H.; Wolfbeis, O. S. Luminescent sensing of oxygen using a quenchable probe and upconverting nanoparticles. Angew. Chem. Int. Ed. 2011, 50, 260–263.

    Article  Google Scholar 

  15. [15]

    Chatterjee, D. K.; Gnanasammandhan, M. K.; Zhang, Y. Small upconverting fluorescent nanoparticles for biomedical applications. Small 2010, 6, 2781–2795.

    Article  Google Scholar 

  16. [16]

    Zhou, J.; Liu, Z.; Li, F. Y. Upconversion nanophosphors for small-animal imaging. Chem. Soc. Rev. 2012, 41, 1323–1349.

    Article  Google Scholar 

  17. [17]

    Haase, M.; Schäfer, H. Upconverting nanoparticles. Angew. Chem. Int. Ed. 2011, 50, 5808–5829.

    Article  Google Scholar 

  18. [18]

    Chen, Y.; Ai, K. L.; Liu, Y. L.; Lu, L. H. Tailor-made charge-conversional nanocomposite for pH-responsive drug delivery and cell imaging. ACS Appl. Mater. Interfaces 2014, 6, 655–663.

    Article  Google Scholar 

  19. [19]

    Yang, D.; Dai, Y.; Liu, J.; Zhou, Y.; Chen, Y.; Li, C.; Ma, P.; Lin, J. Ultra-small BaGdF5-based upconversion nanoparticles as drug carriers and multimodal imaging probes. Biomaterials 2014, 35, 2011–2023.

    Article  Google Scholar 

  20. [20]

    Li, C. X.; Yang, D. M.; Ma, P. A.; Chen, Y. Y.; Wu, Y.; Hou, Z. Y.; Dai, Y. L.; Zhao, J. H.; Sui, C. P.; Lin, J. Multifunctional upconversion mesoporous silica nanostructures for dual modal imaging and in vivo drug delivery. Small 2013, 9, 4150–4159.

    Article  Google Scholar 

  21. [21]

    Wang, C.; Cheng, L.; Liu, Z. Drug delivery with upconversion nanoparticles for multi-functional targeted cancer cell imaging and therapy. Biomaterials 2011, 32, 1110–1120.

    Article  Google Scholar 

  22. [22]

    Das, G. K.; Stark, D. T.; Kennedy, I. M. Potential toxicity of up-converting nanoparticles encapsulated with a bilayer formed by ligand attraction. Langmuir 2014, 30, 8167–8176.

    Article  Google Scholar 

  23. [23]

    Atabaev, T. Sh.; Lee, J. H.; Han, D. W.; Hwang, Y. H.; Kim, H. K. Cytotoxicity and cell imaging potentials of submicron color-tunable yttria particles. J. Biomed. Mater. Res. Part A 2012, 100, 2287–2294.

    Google Scholar 

  24. [24]

    Gao, G.; Zhang, C. L.; Zhou, Z. J.; Zhang, X.; Ma, J. B.; Li, C.; Jin, W. L.; Cui, D. X. One-pot hydrothermal synthesis of lanthanide ions doped one-dimensional upconversion submicrocrystals and their potential application in vivo CT imaging. Nanoscale 2013, 5, 351–362.

    Article  Google Scholar 

  25. [25]

    Gupta, B. K.; Narayanan, T. N.; Vithayathil, S. A.; Lee, Y.; Koshy, S.; Reddy, A. L. M.; Saha, A.; Shanker, V.; Singh, V. N.; Kaipparettu, B. A. et al. Highly luminescent-paramagnetic nanophosphor probes for in vitro high-contrast imaging of human breast cancer cells. Small 2012, 8, 3028–3034.

    Article  Google Scholar 

  26. [26]

    Guo, H. C.; Hao, R. Z.; Qian, H. S.; Sun, S. Q.; Sun, D. H.; Yin, H.; Liu, Z. X.; Liu, X. T. Upconversion nanoparticles modified with aminosilanes as carriers of DNA vaccine for foot-and-mouth disease. Appl. Microbiol. Biotechnol. 2012, 95, 1253–1263.

    Article  Google Scholar 

  27. [27]

    Yang, D. M.; Dai, Y. L.; Ma, P. A.; Kang, X. J.; Cheng, Z. Y.; Li, C. X.; Lin, J. One-step synthesis of small-sized and water-soluble NaREF4 upconversion nanoparticles for in vitro cell imaging and drug delivery. Chem. Eur. J. 2013, 19, 2685–2694.

    Article  Google Scholar 

  28. [28]

    Wang, C.; Cheng, L.; Xu, H.; Liu, Z. Towards whole-body imaging at the single cell level using ultra-sensitive stem cell labeling with oligo-arginine modified upconversion nanoparticles. Biomaterials 2012, 33, 4872–4881.

    Article  Google Scholar 

  29. [29]

    Zhao, L.; Kutikov, A.; Shen, J.; Duan, C. Y.; Song, J.; Han, G. Stem cell labeling using polyethylenimine conjugated (α-NaYbF4:Tm3+)/CaF2 upconversion nanoparticles. Theranostics 2013, 3, 249–257.

    Article  Google Scholar 

  30. [30]

    Abdul Jalil, R.; Zhang, Y. Biocompatibility of silica coated NaYF4 upconversion fluorescent nanocrystals. Biomaterials 2008, 29, 4122–4128.

    Article  Google Scholar 

  31. [31]

    Nadort, A.; Sreenivasan, V. K. A.; Song, Z.; Grebenik, E. A.; Nechaev, A. V.; Semchishen, V. A.; Panchenko, V. Y.; Zvyagin, A. V. Quantitative imaging of single upconversion nanoparticles in biological tissue. PloS One 2013, 8, e63292.

    Article  Google Scholar 

  32. [32]

    Mahajan, S.; Aalinkeel, R.; Reynolds, J.; Nair, B.; Sykes, D.; Law, W. C.; Prasad, P.; Schwartz, S. Innovative nanotherapy for the treatment of the chronic skin condition, rosacea (P3259). J. Immunol. 2013, 190, 192.

    Google Scholar 

  33. [33]

    Chatterjee, D. K.; Rufaihah, A. J.; Zhang, Y. Upconversion fluorescence imaging of cells and small animals using lanthanide doped nanocrystals. Biomaterials 2008, 29, 937–943.

    Article  Google Scholar 

  34. [34]

    Xia, A.; Chen, M.; Gao, Y.; Wu, D.; Feng, W.; Li, F. Gd3+ complex-modified NaLuF4-based upconversion nanophosphors for trimodality imaging of nir-to-nir upconversion luminescence, X-ray computed tomography and magnetic resonance. Biomaterials 2012, 33, 5394–5405.

    Article  Google Scholar 

  35. [35]

    Ma, J. B.; Huang, P.; He, M.; Pan, L. Y.; Zhou, Z. J.; Feng, L. L.; Gao, G.; Cui, D. X. Folic acid-conjugated LaF3:Yb,Tm@SiO2 nanoprobes for targeting dual-modality imaging of upconversion luminescence and X-ray computed tomography. J. Phys. Chem. B 2012, 116, 14062–14070.

    Article  Google Scholar 

  36. [36]

    Zhou, N.; Qiu, P.; Wang, K.; Fu, H. L.; Gao, G.; He, R.; Cui, D. X. Shape-controllable synthesis of hydrophilic NaLuF4:Yb,Er nanocrystals by a surfactant-assistant two-phase system. Nanoscale Res. Lett. 2013, 8, 518.

    Article  Google Scholar 

  37. [37]

    Grebenik, E. A.; Nadort, A.; Generalova, A. N.; Nechaev, A. V.; Sreenivasan, V. K.; Khaydukov, E. V.; Semchishen, V. A.; Popov, A. P.; Sokolov, V. I.; Akhmanov, A. S. et al. Feasibility study of the optical imaging of a breast cancer lesion labeled with upconversion nanoparticle biocomplexes. J. Biomed. Opt. 2013, 18, 076004.

    Article  Google Scholar 

  38. [38]

    Andrade, Â. L.; Fabris, J. D.; Ardisson, J. D.; Valente, M. A.; Ferreira, J. M. F. Effect of tetramethylammonium hydroxide on nucleation, surface modification and growth of magnetic nanoparticles. J. Nanomater. 2012, 2012, 1–10.

    Google Scholar 

  39. [39]

    Generalova, A. N.; Kochneva, I. K.; Khaydukov, E. V.; Semchishen, V. A.; Guller, A. E.; Nechaev, A. V.; Shekhter, A. B.; Zubov, V. P.; Zvyagin, A. V.; Deyev, S. M. Submicron polyacrolein particles in situ embedded with upconversion nanoparticles for bioassay. Nanoscale 2014, DOI: 10.1039/c4nr05908e.

    Google Scholar 

  40. [40]

    Li, M.; Rouaud, O.; Poncelet, D. Microencapsulation by solvent evaporation: State of the art for process engineering approaches. Int. J. Pharm. 2008, 363, 26–39.

    Article  Google Scholar 

  41. [41]

    Bao, Y.; Luu, Q. A. N.; Lin, C. K.; Schloss, J. M.; May, P. S.; Jiang, C. Y. Layer-by-layer assembly of freestanding thin films with homogeneously distributed upconversion nanocrystals. J. Mater. Chem. 2010, 20, 8356–8361.

    Article  Google Scholar 

  42. [42]

    Generalova, A. N.; Oleinikov, V. A.; Zarifullina, M. M.; Lankina, E. V.; Sizova, S. V.; Artemyev, M. V.; Zubov, V. P. Optical sensing quantum dot-labeled polyacrolein particles prepared by layer-by-layer deposition technique. J. Colloid Interface Sci. 2011, 357, 265–272.

    Article  Google Scholar 

  43. [43]

    Boukamp, P.; Petrussevska, R. T.; Breitkreutz, D.; Hornung, J.; Markham, A.; Fusenig, N. E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell Biol. 1988, 106, 761–771.

    Article  Google Scholar 

  44. [44]

    Breitkreutz, D.; Schoop, V. M.; Mirancea, N.; Baur, M.; Stark, H. J.; Fusenig, N. E. Epidermal differentiation and basement membrane formation by hacat cells in surface transplants. Eur. J. Cell Biol. 1998, 75, 273–286.

    Article  Google Scholar 

  45. [45]

    Takashima A. Establishment of fibroblast cultures. Curr. Protoc. Cell Biol. 2001, Chapter 2: Unit 2.1. DOI: 10.1002/0471143030.cb0201s00.

    Google Scholar 

  46. [46]

    Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63.

    Article  Google Scholar 

  47. [47]

    Cao, T. Y.; Yang, Y.; Gao, Y.; Zhou, J.; Li, Z. Q.; Li, F. Y. High-quality water-soluble and surface-functionalized upconversion nanocrystals as luminescent probes for bioimaging. Biomaterials 2011, 32, 2959–2968.

    Article  Google Scholar 

  48. [48]

    Chen, Z. G.; Chen, H. L.; Hu, H.; Yu, M. X.; Li, F. Y.; Zhang, Q.; Zhou, Z. G.; Yi, T.; Huang, C. H. Versatile synthesis strategy for carboxylic acid-functionalized upconverting nanophosphors as biological labels. J. Am. Chem. Soc. 2008, 130, 3023–3029.

    Article  Google Scholar 

  49. [49]

    Bogdan, N.; Vetrone, F.; Ozin, G. A.; Capobianco, J. A. Synthesis of ligand-free colloidally stable water dispersible brightly luminescent lanthanide-doped upconverting nanoparticles. Nano Lett. 2011, 11, 835–840.

    Article  Google Scholar 

  50. [50]

    Bumajdad, A.; Eastoe, J.; Zaki, M. I.; Heenan, R. K.; Pasupulety, L. Generation of metal oxide nanoparticles in optimised microemulsions. J. Colloid Interface Sci. 2007, 312, 68–75.

    Article  Google Scholar 

  51. [51]

    Pellegrino, T.; Manna, L.; Kudera, S.; Liedl, T.; Koktysh, D.; Rogach, A. L.; Keller, S.; Rädler, J.; Natile, G.; Parak, W. J. Hydrophobic nanocrystals coated with an amphiphilic polymer shell: A general route to water soluble nanocrystals. Nano Lett. 2004, 4, 703–707.

    Article  Google Scholar 

  52. [52]

    Jin, J. F.; Gu, Y. J.; Man, C. W. Y.; Cheng, J. P.; Xu, Z. H.; Zhang, Y.; Wang, H. S.; Lee, V. H. Y.; Cheng, S. H.; Wong, W. T. Polymer-coated NaYF4:Yb3+,Er3+ upconversion nanoparticles for charge-dependent cellular imaging. ACS Nano 2011, 5, 7838–7847.

    Article  Google Scholar 

  53. [53]

    Sheftel, V. O.; Kataeva, S. E. Migration of Harmful Chemicals from Polymeric Materials. Khimiya: Moscow, 1978.

    Google Scholar 

  54. [54]

    Forrest, M. L.; Koerber, J. T.; Pack, D. W. A degradable polyethylenimine derivative with low toxicity for highly efficient gene delivery. Bioconjugate Chem. 2003, 14, 934–940.

    Article  Google Scholar 

  55. [55]

    Tiyaboonchai, W.; Woiszwillo, J.; Middaugh, C. R. Formulation and characterization of DNA-polyethylenimine-dextran sulfate nanoparticles. Eur. J. Pharm. Sci. 2003, 19, 191–202.

    Article  Google Scholar 

  56. [56]

    Chen, G. Y.; Qiu, H. L.; Prasad, P. N.; Chen, X. Y. Upconversion nanoparticles: Design, nanochemistry, and applications in theranostics. Chem. Rev. 2014, 114, 5161–5214.

    Article  Google Scholar 

  57. [57]

    Müller, R. H.; Maaßen, S.; Weyhers, H.; Specht, F.; Lucks, J. S. Cytotoxicity of magnetite-loaded polylactide, polylactide/glycolide particles and solid lipid nanoparticles. Int. J. Pharm. 1996, 138, 85–94.

    Article  Google Scholar 

  58. [58]

    Pham, Q. P.; Sharma, U.; Mikos, A. G. Electrospinning of polymeric nanofibers for tissue engineering applications: A review. Tissue Eng. 2006, 12, 1197–1211.

    Article  Google Scholar 

  59. [59]

    Ogawara, K. I.; Yoshida, M.; Takakura, Y.; Hashida, M.; Higaki, K.; Kimura, T. Interaction of polystyrene microspheres with liver cells: Roles of membrane receptors and serum proteins. Biochim. Biophys. Acta 1999, 1472, 165–172.

    Article  Google Scholar 

  60. [60]

    Kelf, T. A.; Sreenivasan, V. K. A.; Sun, J.; Kim, E. J.; Goldys, E. M.; Zvyagin, A. V. Non-specific cellular uptake of surface-functionalized quantum dots. Nanotechnology 2010, 21, 285105.

    Article  Google Scholar 

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Guller, A.E., Generalova, A.N., Petersen, E.V. et al. Cytotoxicity and non-specific cellular uptake of bare and surface-modified upconversion nanoparticles in human skin cells. Nano Res. 8, 1546–1562 (2015).

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  • nanoparticle
  • upconversion
  • surface modification
  • biocompatibility
  • cytotoxicity
  • human skin