Analysis of Stable Chelate-free Gadolinium Loaded Titanium Dioxide Nanoparticles for MRI-Guided Radionuclide Stimulated Cancer Treatment

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. Methods: 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 fluo-rescence. 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 stabi-lized the nanoconstruct and minimized aggregation. The TiO₂-Gd-Tf maintained ROS-generating capabil-ity 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 de-termining 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.