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Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection

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Abstract

Titanium dioxide (TiO2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ posttreatment labeling approaches to stain TiO2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO2 nanoparticles with alkyneconjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.

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Acknowledgements

This research was supported by the National Institutes of Health (Nos. CA107467, EB002100, U54CA119341 and GM104530). Implementation of the Bionanoprobe is supported by NIH ARRA (No. SP0007167). Confocal optical imaging work was performed at the Northwestern University Center for Advanced Microscopy generously supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. Confocal microscopy was performed on a Nikon A1R multiphoton microscope, acquired through the support of NIH 1S10OD010398-01. Work at the Advanced Photon Source at Argonne National Laboratory was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences contract No. DE-AC02-06CH11357. Metal analysis was performed at the Northwestern University Quantitative Bio-element Imaging Center generously supported by NASA Ames Research Center (No. NNA06CB93G). Use of the Simpson Querrey Institute Analytical BioNanoTechnology Equipment Core (ANTEC) facility was supported by the U.S. Army Research Office, the U.S. Army Medical Research and Materiel Command, and Northwestern University funding received from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF NNCI-1542205). Cryo-TEM work was performed at the Northwestern University Biological Imaging Facility by Imaging Specialist Charlene Wilke. The authors thank Dr. Teng-Leong Chew for his valuable discussion and advice.

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Author notes

  1. Ted Thurn

    Present address: Present Address: U.S. Department of State, 2201 C Street, NW, Washington, DC, 20520, USA

  2. William Liu

    Present address: Present Address: Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, Maryland, 20993, USA

  3. Remon Bazak

    Present address: Present Address: Department of Otorhinolaryngology and Head & Neck Surgery, University of Alexandria Medical School, Azarita Medical Campus, Champlollion Street, Khartoum Square, Alexandria, 21547, Egypt

Authors and Affiliations

  1. Department of Radiation Oncology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, 60611, USA

    Koshonna Brown, Ted Thurn, Lun Xin, William Liu, Remon Bazak, Tatjana Paunesku & Gayle E. Woloschak

  2. X-ray Science Division, Advanced Photon Source, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, Illinois, 60439, USA

    Si Chen, Barry Lai & Stefan Vogt

  3. Department of Physics & Astronomy, Weinberg College of Arts and Sciences, 2145 Sheridan Road, Evanston, Illinois, 60208, USA

    Chris Jacobsen

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  1. Koshonna Brown
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Brown, K., Thurn, T., Xin, L. et al. Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection. Nano Res. 11, 464–476 (2018). https://doi.org/10.1007/s12274-017-1654-8

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