Synthesis and in vivo evaluation of PEG-BP–BaYbF5 nanoparticles for computed tomography imaging and their toxicity

Computed tomography (CT) is one of the most widespread imaging techniques in clinical use worldwide. CT contrast agents are administered to improve soft tissue contrast and highlight blood vessels. However, the range of CT contrast agents available in the clinic is limited and they suffer from short-circulation times and low k-edge values that result in the need for high doses for in vivo applications. Nanomaterials containing a mixture of electron-dense elements, such as BaYbF5 nanoparticles, have shown promise as more efficient CT contrast agents, but they require biocompatible coatings for biomedical applications. Here, we explore the use of a bifunctional PEG polymer (5 kDa) containing a terminal bisphosphonate (BP) anchor for efficient binding to the surface of BaYbF5 nanomaterials. The resulting PEG(5)-BP–BaYbF5 nanoparticles were synthesized and characterized using TEM, DLS, TGA, XRD and Z-potential measurements. Their in vitro stability was verified and their ability to produce CT contrast in a wide range of X-ray energies, covering preclinical and clinical scanners, was demonstrated. In vitro toxicity studies with PEG(5)-BP–BaYbF5 in the phagocytic pro-monocytic human cell line U937 did not identify toxic effects, even at high concentrations (30 mM). In vivo, PEG(5)-BP–BaYbF5 exhibited efficient CT contrast for angiography imaging, highlighting blood vessels and vascular organs, and long circulation times as expected from the PEG coating. However, at late time points (48 h), in vivo toxicity was observed. While the causes could not be completely elucidated, in vitro studies suggest that decomposition and release of Yb3+ and/or Ba2+ metal ions after decomposition of PEG(5)-BP–BaYbF5 may play a role. Overall, despite the promising CT contrast properties, our results suggest that BaYbF5 nanomaterials may suffer from significant long-term toxicities.


S3. PEG(5)-BP-BaYbF5 PEG density calculations
Calculations of the number of PEG(5)-BP molecules bound to each NP were performed assuming the NPs are spheres of a 9.5 nm diameter resulting in a NP volume of 4.49 x 10 -19 cm 3 , and composed of BaYbF5. Density of BaYBF5 was estimated to be the weighted average of YbF3 and BaF2 density (6.55 g/cm 3 ). Based on these assumptions, the mass of a NP BaYbF5 core was estimated to be 2.94 x10 -18 g.
Hence, the number of PEG molecules per nanoparticle is calculated to be 196.

Calculation of theoretical maximum density of PEG(5)-BP/NPs
The surface area of a 9.5 nm sphere is 283.4 nm 2 . The footprint of a BP is approximately 0.85 nm2 (calculated from a computational model using Chem3D, Cambridge Software). Using these values, the theoretical maximum number of BPs that can bind to a 9.5 nm sphere is 333.

Maximum number of PEG(5)-BP/NPs calculated using the amount of non-bound PEG(5)-BP after purification
Based on a scaled up version of the reaction in which 40 mg of OA-BaYBF5 Nanoparticles were reacted with 80 mg of PEG(5)-BP.

-Calculation of number of Yb atoms/NPs
The number of Yb atoms in a nanoparticle is equal to the number of BaYbF5 units. This can be calculated as (NPs mass x Nav / MW BaYbF5 = 2.94 x10 -18 g x 6.022 x10 23 atoms/mol /405.32 g/mol) Resulting in 4366.5 Yb atoms/NP

-Calculation of number of BaYBF5 NPs
The Yb concentration of the PEG(5)-BP-BaYBF5 dispersion was measured to be 24.55 mM by ICP-MS, which corresponds to 9.82x 10 -5 moles and 5.19 x 10 19 Yb Atoms in 4 mL.

-Calculation of number of PEG molecules bound to BaYBF5 NPs
Using the mass of the non-bound PEG(5)-BP, obtained from the washings during purification of the pegylated nanoparticles allowed us to calculate that a maximum of 46.5 mg of PEG(5)-BP was bound to the BaYBF5 NPs.
Hence, using the average MW of PEG(5)-BP value of 5307 g/mol, we can calculate that this corresponds to a maximum of approximately 5.28 x 10 18 PEG(5)-BP molecules.

-Calculation of number of PEGs/NPs
The maximum number of PEG molecules per NPs is calculated to be 5.28 x 10 18 / 1.35 x 10 16 = 389.6 PEG(5)-BP/NP Since the maximum PEG(5)-BP/NPs density calculated using the recovered PEG(5)-BP was higher than the maximum theoretical density, the latter was deemed more accurate and used for comparison with value obtained from TGA measurements. Based on these value the density of PEG(5)-BP molecules on the surface of the NPs was 58.85% of the maximum theoretical density, which is consistent with the PEG moieties being arranged in a brush regime.