Ultrasmall cerium oxide nanoparticles as highly sensitive X-ray contrast agents and their antioxidant effect

Abstract

Owing to their theranostic properties, cerium oxide (CeO₂) nanoparticles have attracted considerable attention for their key applications in nanomedicine. In this study, ultrasmall CeO₂ nanoparticles (particle diameter = 1-3 nm) as X-ray contrast agents with an antioxidant effect were investigated for the first time. The nanoparticles were coated with hydrophilic and biocompatible poly(acrylic acid) (PAA) and poly(acrylic acid-co-maleic acid) (PAAMA) to ensure satisfactory colloidal stability in aqueous media and low cellular toxicity. The synthesized nanoparticles were characterized using high-resolution transmission electron microscopy, X-ray diffraction, Fourier transform-infrared spectroscopy, thermogravimetric analysis, dynamic light scattering, cell viability assay, photoluminescence spectroscopy, and X-ray computed tomography (CT). Their potential as X-ray contrast agents was demonstrated by measuring phantom images and in vivo CT images in mice injected intravenously and intraperitoneally. The X-ray attenuation of these nanoparticles was greater than that of the commercial X-ray contrast agent Ultravist and those of larger CeO₂ nanoparticles reported previously. In addition, they exhibited an antioxidant effect for the removal of hydrogen peroxide. The results confirmed that the PAA- and PAAMA-coated ultrasmall CeO₂ nanoparticles demonstrate potential as highly sensitive radioprotective or theranostic X-ray contrast agents.

Fig. 1. (a) Photographs of PAA- and PAAMA-coated ultrasmall CeO₂ nanoparticles dispersed in aqueous media and water. (b) Zeta potential (ζ) curves and Gaussian function fits to obtain ζ_avg.

Fig. 2. (a(i)), (a(ii)), (b(i)), and (b(ii)) HRTEM images: nanoparticles enclosed within the dotted circles in (b(i)) and (b(ii)) were magnified as indicated by the arrows (scale bar = 2 nm). (c(i)) and (c(ii)) HAADF-STEM images. (d(i)) and (d(ii)) Elemental mapping in the HAADF-STEM mode. (e) Particle diameter distributions and log–normal function fits to obtain d_avg. (f) DLS patterns and log–normal function fits to obtain a_avg. In (a)–(d), (i) indicates PAA-coated ultrasmall CeO₂ nanoparticles and (ii) indicates PAAMA-coated ultrasmall CeO₂ nanoparticles.

Fig. 3. XRD patterns of the powder samples of the PAA- and PAAMA-coated ultrasmall CeO₂ nanoparticles (a) before and (b) after TGA up to 900 °C under airflow. The peaks at the top of the peaks are (hkl) Miller indices of bulk CeO₂ with an FCC crystal structure.

Fig. 4. FT-IR absorption spectra of (a) free PAA and PAA-coated ultrasmall CeO₂ nanoparticles and (b) free PAAMA and PAAMA-coated ultrasmall CeO₂ nanoparticles. “as” and “ss” indicate the antisymmetric and symmetric stretching vibrations of COO⁻, respectively. (c) TGA curves of the PAA- and PAAMA-coated ultrasmall CeO₂ nanoparticles under air flow. (d) Schematic of the coating structures of PAA and PAAMA polymers on the nanoparticle surfaces via electrostatic (i.e., hard acid–base) bonding between the COO⁻ groups of the polymers and Ce⁴⁺ on the nanoparticle surfaces (the minor Ce³⁺ ions also exist on the nanoparticle surfaces, but only the major Ce⁴⁺ ions were displayed on the nanoparticle surfaces).

Fig. 5. In vitro cell viability of (a) NCTC1469 and (b) DU145 cells and optical microscopy images of (c) NCTC1469 and (d) DU145 cells 48 h after incubation with the PAA- and PAAMA-coated ultrasmall CeO₂ nanoparticles up to 500 μM [Ce]. Scale bar = 70 nm.

Fig. 6. Photographs of various solutions up to 24 h: (a) 0.01 mM Rh B, (b) 0.1% H₂O₂, (c) PAA- and (d) PAAMA-coated ultrasmall CeO₂ nanoparticles dispersed in aqueous media (0.1 mM [Ce]), (e) 0.01 mM Rh B + 0.05% H₂O₂, (f) 0.01 mM Rh B + PAA-coated ultrasmall CeO₂ nanoparticles (0.05 mM [Ce]), (g) 0.01 mM Rh B + PAAMA-coated ultrasmall CeO₂ nanoparticles (0.05 mM [Ce]), (h) 0.01 mM Rh B + 0.05% H₂O₂ + PAA-coated ultrasmall CeO₂ nanoparticles (0.05 mM [Ce]), (i) 0.01 mM Rh B + 0.05% H₂O₂ + PAAMA-coated ultrasmall CeO₂ nanoparticles (0.05 mM [Ce]). U = 365 nm UV irradiation (power = 15 W) and N = no UV irradiation.

Fig. 7. PL spectra of (a) solution-a (i.e., 0.01 mM Rh B), (b) solution-e (i.e., 0.01 mM Rh B + 0.05% H₂O₂), (c) solution-f {i.e., 0.01 mM Rh B + PAA-coated ultrasmall CeO₂ nanoparticles (0.05 mM [Ce])}, (d) solution-g {i.e., 0.01 mM Rh B + PAAMA-coated ultrasmall CeO₂ nanoparticles (0.05 mM [Ce])}, (e) solution-h {i.e., 0.01 mM Rh B + 0.05% H₂O₂ + PAA-coated ultrasmall CeO₂ nanoparticles (0.05 mM [Ce])}, (f) solution-i {i.e., 0.01 mM Rh B + 0.05% H₂O₂ + PAAMA-coated ultrasmall CeO₂ nanoparticles (0.05 mM [Ce])} in Fig. 6: U = 365 nm UV irradiation and N = no UV irradiation. (g) Plots of Rh B degradation efficiency (%) for solutions-a, -e, -f, -g, -h, and -i in Fig. 6.

Fig. 8. (a) X-ray phantom images of Ultravist and PAA- and PAAMA-coated ultrasmall CeO₂ nanoparticles dispersed in aqueous media at an X-ray source voltage of 70 kV_p. (b) Plot of the linear attenuation coefficients of Ce and I versus radiation photon energy. Plots of the X-ray attenuation as a function of the (c) atomic concentrations of [Ce] and [I] and (d) number density of the nanoparticles and Ultravist: slopes of the dotted lines correspond to X-ray attenuation efficiencies (η). (e) Comparison of η values: dextran-coated CeO₂ nanoparticles (d = 4.8 nm, 80 kV_p), porous Ce₂(CO₃)₂O·H₂O nanoparticles (d = 196.6 nm, 80 kV_p), and polymer-coated ultrasmall CeO₂ nanoparticles [d = (1.8 + 2.0)/2 = 1.9 nm, 70 kV_p] (this study). Water: 0 HU.

Fig. 9. (a) In vivo CT images of the mice bladder before and after intravenous (IV) and intraperitoneal (IP) injections of an aqueous suspension sample of PAA-coated ultrasmall CeO₂ nanoparticles at 70 kV_p. The dotted circles at the bladder indicate the region of interest (ROI). (b) Contrast plots of the SNR-ROI of the bladder as a function of time.