HL-K6-3512) as well as by grants (HL-02212 and HL-16346) from the National Institutes of Health. A Fulbright Travelling Fellowship to C. G. C. is gratefully acknowledged.

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Nanomaterials have been shown to have physical and chemical properties that have opened new avenues for cancer diagnosis and therapy. Nanoconstructs that enhance existing treatments for cancer, such as radiation therapy, are being explored in several different ways. Two general paths toward nanomaterial-enabled radiosensitization have been explored: (1) improving the effectiveness of ionizing radiation and (2) modulating cellular pathways leading to a disturbance of cellular homeostasis, thus rendering the cells more susceptible to radiation-induced damage. A variety of different agents that work via one of these two approaches have been explored, many of which modulate direct and indirect DNA damage (gold), radiosensitivity through hyperthermia (Fe), and different cellular pathways. There have been many in vitro successes with the use of nanomaterials for radiosensitization, but in vivo testing has been less efficacious, predominantly because of difficulty in targeting the nanoparticles. As improved methods for tumor targeting become available, it is anticipated that nanomaterials can become clinically useful radiosensitizers for radiation therapy.

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 post-treatment 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 alkyne-conjugated 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.