Prakash Nallathamby1 Viktoriya Sokolova2 Oleg Prymak2 Matthias Epple2 Ryan Roeder1

1, University of Notre Dame, Notre Dame, Indiana, United States
2, University of Duisburg-Essen, Duisburg, , Germany

Hafnium oxide (HfO2) with a k-edge of 50 keV is being explored as a clinical X-ray contrast agent. HfO2 NPs are also in clinical trials as radiosensitizers that induce immune action against tumor sites. However, the unpredictable stability of commercially available HfO2 makes it hard to predict their pharmacokinetics and their size range of ((50-100 nm) results in zero clearance from an in vivo system. Therefore, in this study we have executed a modular approach for the design and scalable synthesis of novel, non-sintered, 4-8 nm metal oxide nanoparticles (e.g. Hafnium oxide, Gadolinium oxide) with 40-60 fold higher X-ray attenuation cross-sections than the NPs diameter. Contrast-enhanced computed tomography (CT) and spectral (color) X-ray CT with the aid of these new class of probes have the potential to enable targeted image guided therapeutics with CT as a lower cost and higher resolution alternative to PET and MRI.
Amorphous HfO2 seed NPs, ~1.5 nm in size, embedded in a polycationic mesh and immobilized inside a silica template, were prepared by the simultaneous reduction and stabilization of tetrakis dimethyl amido hafnium by oleylamine at >250°C in a polyol solvent. Seed NPs were subsequently subjected to high-temperature calcination (>650°C), followed by alkaline digestion of the silica template, to prepare crystalline HfO2 NPs. As-prepared HfO2 NPs were surface functionalized with a monolayer of fluorescent silane to enable fluorescence imaging. The hydrodynamic diameter and zeta potential were measured using dynamic light scattering. In vitro cytocompatibility was characterized using established methods which included Live/Dead assay and confocal laser scanning microscopy after incubating NPs with HeLa cancer cells and THP-1 macrophage cells at 25-200 mg NPs/100,000 cells
We achieved >95% efficient conversion of the amorphous seed NPs to crystalline orthorhombic-HfO2 NPs. HfO2 NPs were ~4-5 nm in diameter and formed non-sintered flocculants, 220-290 nm in diameter. As-prepared CY5 tagged-HfO2 NPs exhibited simultaneous X-ray contrast and fluorescence in multispectral imaging. Both HfO2 and CY5 tagged-HfO2 NPs remained well-dispersed over 24 h. The measured zeta potential was -13 to -16 mV. Encapsulating the HfO2 NPs in a SiO2 shell reduced the hydrodynamic diameter to ~30 nm and inhibited the formation of flocculants. MTT and Live/Dead assays confirmed that the HfO2 NPs were neither cytotoxic nor pro-inflammatory. Confocal microscopy confirmed highly efficient uptake of Cy5-HfO2 NPs by HeLa cells and THP-1 cells.
HfO2 NPs were prepared using a novel templated synthesis resulting in crystalline NPs, ~4-5 nm in diameter, which flocculate into 220-290 nm clusters. Therefore, these NPs provide both long blood circulation and eventual renal clearance through phagocytic breakdown after delivery as radiographic imaging probe or radiosensitizer.