A novel synthetic approach of cerium oxide nanoparticles with improved biomedical activity

Abstract

Cerium oxide nanoparticles (CNPs) are novel synthetic antioxidant agents proposed for treating oxidative stress-related diseases. The synthesis of high-quality CNPs for biomedical applications remains a challenging task. A major concern for a safe use of CNPs as pharmacological agents is their tendency to agglomerate. Herein we present a simple direct precipitation approach, exploiting ethylene glycol as synthesis co-factor, to synthesize at room temperature nanocrystalline sub-10 nm CNPs, followed by a surface silanization approach to improve nanoparticle dispersibility in biological fluids. CNPs were characterized using transmission electron microscopy (TEM) observations, X-ray diffraction (XRD) analysis, thermogravimetric analysis (TGA), Fourier-transform infrared (FT-IR) spectroscopy, proton nuclear magnetic resonance (¹H-NMR) spectroscopy, dynamic light scattering (DLS) and zeta potential measurements. CNP redox activity was studied in abiotic systems using electron spin resonance (ESR) measurements, and in vitro on human cell models. In-situ silanization improved CNP colloidal stability, in comparison with non-functionalized particles, and allowed at the same time improving their original biological activity, yielding thus functionalized CNPs suitable for biomedical applications.

Figure 1

Physico-chemical characterization of C1 nanoparticles. (a) XRD diffraction patterns and Miller indexes of C1. (b) TEM image. (c) TGA curve. (d) FTIR absorbance spectra with the inset of the CH₂ stretching zone.

Figure 2

Physico-chemical characterization of C2 nanoparticles. (a) XRD diffraction patterns and Miller indexes. (b) TEM image. (c) TGA curve. (d) FTIR absorbance spectra; the inset shows the CH₂ stretching zone.

Figure 3

C2 nanoparticle surface silanization. (a) TGA analysis of C2, C3 and C4 samples. (b) FTIR absorbance spectra. (c) ¹H-NMR spectra of C3 and C4 samples. The peaks were assigned to the APTES and MEEETES molecular structures.

Figure 4

CNP colloidal stability over time. C1, C2, and C3 sample residual concentration into the supernatant after 7 and 14 days.

Figure 5

Abiotic analysis of CNP redox activity. (a) ESR spectra of DMPO spin abduct signals produced by UV-irradiated TiO₂ suspensions at 800 µg/mL ± CNPs at 400 µg/mL. On the right a table reporting the DMPO-OH residual signal in the presence of C1, C1–450, C2, C3, and C4 samples. (b) ESR analysis of TEMPOL decomposition into TEMPONE during continuous irradiation of TiO₂ (800 µg/mL) ± CNPs (400 µg/mL). On the left the TEMPOL signal reduction and the TEMPONE formation kinetics for 525 s, on the right % of TEMPOL disappearance and TEMPONE formation after 525 s.

Figure 6

CNP nanoparticles do not show any toxicity. (a) Time course of cellular proliferation for 12–72 h measured by cells counting reported as number of cells. (b) Basal ROS levels measured by DHR fluorescent signal detected by flow cytometry 24 h after cell treatment with CNPs (200 μg/mL). Values are the mean of ≥3 independent experiments ± SD; *p < 0.05 (ANOVA). Significance with respect to the control group is shown.

Figure 7

Redox active CNPs protect cells from hydrogen peroxide and UVB. (a) ROS levels increment measured by DHR fluorescent signal detected by flow cytometry 1 h after cell treatment with H₂O₂ ± CNPs. (b) % of apoptosis detected 24 h after cell treatment with H₂O₂ ± CNPs. (c) % of apoptosis detected 24 h after cell treatment with UVB ± CNPs. Values are the mean of ≥3 independent experiments ± SD; *p < 0.05 (ANOVA). Significance with respect to the control group is shown. In (b) and (c) also the significance with respect to the C3 group is shown, ^#p < 0.05 (ANOVA).

Figure 8

CNP redox activity over time. % protection from apoptosis in cells treated with H₂O₂ ± C1, C2, and C3 supernatant suspensions after 7 and 14 days. Values are the mean of ≥3 independent experiments ± SD.