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Background: Recent studies demonstrate that titanium dioxide nanoparticles (TiO2 NPs) are an effective source of reactive oxygen species (ROS) for photodynamic therapy and radionuclide stimulated dynamic therapy (RaST). Unfortunately, tracking the in vivo distribution of TiO2 NPs noninvasively remains elusive. Objective: Given the use of gadolinium (Gd) chelates as effective contrast agents for magnetic resonance imaging (MRI), this study aims to (1) develop hybrid TiO2-Gd NPs that exhibit high relaxivity for tracking the NPs without loss of ROS generating capacity; and (2) establish a simple colorimetric assay for quantifying Gd loading and stability. Method: A chelate-free, heat-induced method was used to load Gd onto TiO2 NPs, which was coated with transferrin (Tf). A sensitive colorimetric assay and inductively coupled plasma mass spectrometry (ICP-MS) were used to determine Gd loading and stability of the TiO2-Gd-Tf NPs. Measurement of the relaxivity was performed on a 1.4 T relaxometer and a 4.7 T small animal magnetic resonance scanner to estimate the effects of magnetic field strength. ROS was quantified by activated dichlorodihydrofluorescein diacetate fluorescence. Cell uptake of the NPs and RaST were monitored by fluorescence microscopy. Both 3 T and 4.7 T scanners were used to image the in vivo distribution of intravenously injected NPs in tumor-bearing mice. Results: A simple colorimetric assay accurately determined both the loading and stability of the NPs compared with the expensive and complex ICP-MS method. Coating of the TiO2-Gd NPs with Tf stabilized the nanoconstruct and minimized aggregation. The TiO2-Gd-Tf maintained ROS-generating capability without inducing cell death at a wide range of concentrations but induced significant cell death under RaST conditions in the presence of F-18 radiolabeled 2-fluorodeoxyglucose. The longitudinal (r1 = 10.43 mM-1s-1) and transverse (r2 = 13.43 mM-1s-1) relaxivity of TiO2-Gd-Tf NPs were about twice and thrice, respectively, those of clinically used Gd contrast agent (Gd-DTPA; r1 = 3.77 mM-1s-1 and r2 = 5.51 mM-1s-1) at 1.4 T. While the r1 (8.13 mM-1s-1) reduced to about twice that of Gd-DTPA (4.89 mM-1s-1) at 4.7 T, the corresponding r2 (87.15 mM-1s-1) increased by a factor 22.6 compared to Gd-DTPA (r2 = 3.85). MRI of tumor-bearing mice injected with TiO2-Gd-Tf NPs tracked the NPs distribution and accumulation in tumors. Conclusion: This work demonstrates that Arsenazo III colorimetric assay can substitute ICP-MS for determining the loading and stability of Gd-doped TiO2 NPs. The new nanoconstruct enabled RaST effect in cells, exhibited high relaxivity, and enhanced MRI contrast in tumors in vivo, paving the way for in vivo MRI-guided RaST.

We have developed a generic approach to determine enzyme activities in vitroand monitor their functional status in vivo. Specifically, a method to generate donor (CbOH)−acceptor (Me2NCp) near-infrared (NIR) fluorescent dye pairs for preparing enzyme activatable molecular systems were developed based on the structural template of heptamethine cyanine dyes. Using caspase-3 as a model enzyme, we prepared two new caspase-3 sensitive compounds with high fluorescence quenching efficiency: Me2NCp-DEVD-K(CbOH)-OH (4) and AcGK(Me2NCp)-DEVD-APK(CbOH)-NH2(5). The mechanism of quenching was based on combined effects of direct (classical) and reverse fluorescence resonance energy transfer (FRET). Caspase-3 cleavage of the scissile DEVD amide bond regenerated the NIR fluorescence of both donor and acceptor dyes. While both compounds were cleaved by caspase-3, substrate 5was cleaved more readily than 4, yielding kcatand KM, values of 1.02 ± 0.06 s−1and 15 ± 3 μM, respectively. Treatment of A549 tumor cells with paclitaxel resulted in >2-fold increase in the fluorescence intensity by NIR confocal microscopy, suggesting the activation of pro-caspase-3 to caspase-3. A similar trend was observed in a mouse model, where the fluorescence intensity was nearly twice the value in caspase-3-rich tissue relative to the control. These results demonstrate the use of the same NIR activatable molecular systems for monitoring the activities of enzymes across a wide spatial scale ranging from in vitrokinetics measurements to in celluloand in vivolocalization of caspase-3 activation. The NIR activatable molecular probes provide an effective strategy to screen new drugs in vitroand monitor treatment response in living organisms.