Effects of core titanium crystal dimension and crystal phase on ROS generation and tumour accumulation of transferrin coated titanium dioxide nanoaggregates

Abstract

Radionuclide-stimulated therapy (RaST), which is enhanced by Cherenkov radiation, has enabled deep tissue stimulation of UV photosensitizers, providing a new path for cancer treatment. Previous reports have shown UV-active titanium dioxide (TiO₂) nanoparticles (NPs) modified with transferrin inhibit tumour growth after orthogonal treatment with Cherenkov radiation-emitting radionuclides such as ¹⁸F-fluorodeoxyglucose (FDG). However, poor understanding of TiO₂ NP parameters on reactive oxygen species (ROS) generation and particle distribution limits effective therapy. Here we sought to delineate the effects of crystal phase and core TiO₂ crystal dimension (cTd) on ROS production and particle morphology. We prepared Transferrin (Tf)-TiO₂ nanoaggregates (NAGs) using solvothermally synthesized cTd sizes from 5 to 1000 nm diameter and holo- or apo-transferrin. Holo-transferrin was unable to stabilize TiO₂ NPs while apo-transferrin stabilized TiO₂ into uniform nanoaggregates (NAGs), which were invariant with differing cTd, averaging 116 ± 1.04 nm for cTds below 100 nm. ROS production increased from 5 to 25 nm cTd, attaining a peak at 25 nm before decreasing with larger sizes. The supra-25 nm ROS production decrease was partially driven by a ~1/r ³ surface area decline. Additionally, amorphous TiO₂ of equal core size exhibited a 2.6-fold increase in ROS production compared to anatase NAGs, although limited stability halted further use. Although both 5 and 25 nm anatase cTds formed similarly sized NAGs, 5 nm anatase showed a four-fold higher tumour-to-muscle ratio than the 25 nm NPs in tumour-bearing mice, demonstrating the intricate relationships between physical and biological properties of NAGs. The combined in vivo and ROS results demonstrate that anatase crystals and cTd size of 25 nm or less are ideal particle parameters to balance biodistribution with ROS production efficiency.

Fig. 1. The use of differing core TiO₂ crystal diameter from 5–100 nm results in NAGs of equal size. (A) A representation of the differing core sizes being coated to form regular NAGs of ∼120 nm. (B) Uncoated 25 nm anatase TiO₂ compared against (C) uranyl acetate stained Tf–TiO₂. (D) Histogram of the size distribution of stained 25 nm cTd NCs. (E) X-ray diffraction pattern from the TiO₂ amorphous, rutile and anatase cTds. It is clear the amorphous particles are a mix of anatase, rutile and unstructured crystal domains when referenced against the JCPDS standard XRD card (88-1175 and 84-1286).

Fig. 2. (A) Effects of cTd and crystal structure on the final Tf–TiO₂ particle size and PDI by DLS. 200 nm particles were not filtered due to the proximity to our filter cutoff resulting in low concentration. (B) Band gap energies of bare cTds of TiO₂ calculated from a Tauc plot (Horiba integrating quanta-phi scatter sphere). BaTiO₃ sample was included as calibration, its published E_g is 2.2 eV. TEM of (C) 5, (D) 15, (E) 50 and (F) 200 nm uncoated cTds.

Fig. 3. Stability of 25 nm Tf–TiO₂ particles in water over the course of two months as determined by DLS. The experiment endpoint was a PDI above 0.25.

Fig. 4. (A) Schematic representation of the primary ROS generation by TiO₂ for electrons and holes. Hydroxyl radical production is the primary route sensed by HPF in this work. ROS production rate as quantified through DCF and HPF with changing size (B) and crystal structure (C). The rates were determined through pseudo-first order approximation on the fluorescent curves.

Fig. 5. In vivo biodistribution of two core sizes of Tf–TiO₂ determined by ICP-MS of homogenized tissue. This was done in 4T1 BALB/c mouse models after injecting 200 mg of TiO₂. *P < 0.05.