GdVO4:Eu3+ and LaVO4:Eu3+ Nanoparticles Exacerbate Oxidative Stress in L929 Cells: Potential Implications for Cancer Therapy

Abstract

The therapeutic potential of redox-active nanoscale materials as antioxidant- or reactive oxygen species (ROS)-inducing agents was intensely studied. Herein, we demonstrate that the synthesized and characterized GdVO₄:Eu³⁺ and LaVO₄:Eu³⁺ nanoparticles, which have been already shown to have redox-active, anti-inflammatory, antibacterial, and wound healing properties, both in vitro and in vivo, worsen oxidative stress of L929 cells triggered by hydrogen peroxide or tert-butyl hydroperoxide (tBuOOH) at the concentrations that are safe for intact L929 cells. This effect was observed upon internalization of the investigated nanosized materials and is associated with the cleavage of caspase-3 and caspase-9 without recruitment of caspase-8. Such changes in the caspase cascade indicate activation of the intrinsic caspase-9-dependent mitochondrial but not the extrinsic death, receptor-mediated, and caspase-8-dependent apoptotic pathway. The GdVO₄:Eu³⁺ and LaVO₄:Eu³⁺ nanoparticle-induced apoptosis of oxidatively compromised L929 cells is mediated by ROS overgeneration, Ca²⁺ overload, endoplasmic reticulum stress-associated JNK (c-Jun N-terminal kinase), and DNA damage-inducible transcript 3 (DDIT3). Our findings demonstrate that GdVO₄:Eu³⁺ and LaVO₄:Eu³⁺ nanoparticles aggravate the oxidative stress-induced damage to L929 cells, indicating that they might potentially be applied as anti-cancer agents.

Keywords: apoptosis; caspase; intrinsic apoptosis; nanoparticles; nanotoxicity; oxidative stress.

Figure 1

TEM images of the GdVO₄:Eu³⁺ (Panel (a)) and LaVO₄:Eu³⁺ (Panel (b)) nanoparticles (the insets show the size distribution diagrams and high-resolution micrographs of the synthesized nanoparticles). EDS spectra of the synthesized nanoparticles (Panel (c)). Experimental SAXS curves for the GdVO₄:Eu³⁺ and LaVO₄:Eu³⁺ nanosized materials from the as-prepared aqueous solutions (Panel (d)). The solid lines show the best fit to both the ellipsoidal and cylinder model.

Figure 2

Representative LSCM images demonstrate the uptake of the GdVO₄Eu³⁺ and LaVO₄Eu³⁺ nanoparticles at 20 mg/L by the L929 cells. Internalization of the studies nanoscale materials was quantified (rfu per cell) and revealed a time-dependent pattern. The presented linear velocity (v) indicates the change in the fluorescence intensity of NPs over 1 minute and is calculated based on a linear range of curves (dashed line). ANOVA and post hoc Bonferroni tests were conducted, and the mean ± SD (n = 24) was determined. Note: * (p < 0.05); ** (p < 0.01); and *** (p < 0.001) were compared with the control samples.

Figure 3

Representative LSCM images of the endosome-positive L929 cells following treatment with GdVO₄Eu³⁺ and LaVO₄Eu³⁺ nanoparticles at 20 mg/L for 1 h. The L929 cells were transfected with GFP-Rab5a/GFP-Rab7a and, 24 h after transfection, the cells were exposed to the investigated NPs. The Rab5a and Rab7a proteins were markers of the early and late endosomes, respectively. The GFP-Rab5a/GFP-Rab7a-positive endosomes (green) and NPs’ autofluorescence signals (magenta) were detected. The cell nuclei are shown in blue (DAPI staining). Single-optic section phase contrast and fluorescence-merged imaging (Panel (a)); magnified single-optic section fluorescence imaging of the ROI1 (Panel (b)); orthogonal XZ and YZ projections of the ROI1 (Panel (c)); 3D reconstruction of the ROI1 (Panel (d)); complete 3D reconstruction of the endosomes without and with the NPs’ fluorescence channel (magenta) of the ROI2 (Panel (e)); cross section of the endosomes (red frame) demonstrating the intra-endosomal localization of the NPs (magenta) of the ROI2 (Panel (f)); and pseudocolored and phase-contrast imaging of the endosomal pH using the LysoSensor ratiometric probe (molecular probes, L22460) of the ROI1 (Panels (g,h)). Ratiometric pseudocolored images were constructed from two emission images at 450 ± 33 nm and 510 ± 20 nm, respectively. Both were excited at 365 ± 8 nm. The cells were preliminarily exposed to pH calibration buffers (pH 4.5–6.5).

Figure 4

Confocal microscopy images with an orthogonal projection and 3D reconstruction of the L929 cells treated with the GdVO₄Eu³⁺ and LaVO₄Eu³⁺ nanoparticles at 20 mg/L for 60 min: orthogonal projection (white and red lines) of the cells with highly condensed (Chromatin Condensation Index, CCI = 17.2 ± 4.9) heterochromatin (Panel (a)); orthogonal projection (white and red lines) of the cells with regularly condensed (CCI = 6.8 ± 2.5) heterochromatin (Panel (b)); complete 3D reconstruction of the cells not showing (Panel (c)) and showing (Panel (d)) the channel of nanomaterials-dependent fluorescence (magenta); and cross section of the cell nuclei (red frame) (Panel (e)). Cell nuclei are shown in blue (DAPI staining).

Figure 5

The rare-earth orthovanadate nanoparticles enhanced H₂O₂-mediated oxidative stress in the L929 cells. The GdVO₄:Eu³⁺ nanoparticles increased the DCF-mediated fluorescence in the H₂O₂-treated samples (3 μM), indicating an increase in the ROS production in the L929 cells (Panels (a,b)). Likewise, the LaVO₄:Eu³⁺ nanoparticles promoted H₂O₂-induced redox imbalance in the L929 cells (Panels (c,d)). At the same time, exposure of the L929 cells to both nanoscale materials used does not impair redox homeostasis. ANOVA and post hoc Bonferroni tests were conducted, and the Me and IQR (n = 3) were determined. Note: ** (p < 0.01); and *** (p < 0.001) were compared with the control samples. # (p < 0.05); were compared with the H₂O₂-treated samples.

Figure 6

The GdVO₄:Eu³⁺ and LaVO₄:Eu³⁺ nanoparticles aggravated tBuOOH-induced impairment of redox homeostasis in the L929 cells. The GdVO₄:Eu³⁺ (Panels (a,b)) and LaVO₄:Eu³⁺ nanoparticles (Panels (c,d)) enhanced ROS generation in the L929 cells exposed to tBuOOH (0.5 mM). Data were statistically processed using ANOVA and post hoc Bonferroni tests, and the Me and IQR (n = 3) were determined. Note: ** (p < 0.01); and *** (p < 0.001) were compared with the control samples. # (p < 0.05); ## (p < 0.01) were compared with the tBuOOH-treated samples.

Figure 7

The GdVO₄:Eu³⁺ and LaVO₄:Eu³⁺ nanoparticles triggered Ca²⁺ elevation in the oxidatively damaged L929 cells. The GdVO₄:Eu³⁺ (Panels (a,c)) and LaVO₄:Eu³⁺ nanoparticles (Panels (b,d)) affected the calcium-specific fluorescence intensity in the L929 cells exposed to H₂O₂ and tBuOOH, respectively. ANOVA and Tukey’s tests were conducted, and the mean ± SEM (n = 3) were determined. Note: * (p < 0.05); ** (p < 0.01); and *** (p < 0.001) were compared with the control samples. # (p < 0.05); ## (p < 0.01); and ### (p < 0.001) were compared with the H₂O₂- or tBuOOH-treated samples. Rfu—relative fluorescence units.

Figure 8

The GdVO₄:Eu³⁺ and LaVO₄:Eu³⁺ nanoparticle-induced damage to oxidatively stressed L929 cells was mediated by DDIT3. The GdVO₄:Eu³⁺ (Panels (a,c)) and LaVO₄:Eu³⁺ nanoparticles (Panels (b,d)) increased the DDIT3-specific absorbance in the L929 cells exposed to H₂O₂ and tBuOOH, respectively. ANOVA and Tukey’s tests were conducted, and the mean ± SEM (n = 3) were determined. Note: * (p < 0.05); ** (p < 0.01); and *** (p < 0.001) were compared with the control samples. # (p < 0.05); ## (p < 0.01) were compared with the H₂O₂- or tBuOOH-treated samples. A.u.—arbitrary units.

Figure 9

LSCM representative images of the caspase-3-, caspase-8-, and caspase-9-specific fluorescence in the L929 cells after incubation with the GdVO₄Eu³⁺ (Panel (a)) or LaVO₄Eu³⁺ (Panel (b)) nanoparticles in the presence of H₂O₂. Scale bar is 20 µm.

Figure 10

Representative confocal microscopy images show caspase-3-, caspase-8-, and caspase-9-specific fluorescence in tBuOOH-treated L929 cells exposed to GdVO₄Eu³⁺ (panel (a)) or LaVO₄Eu³⁺ (panel (b)) nanoparticles. Scale bar is 20 µm.