ROS-responsive nano-drug delivery system combining mitochondria-targeting ceria nanoparticles with atorvastatin for acute kidney injury

Abstract

Acute kidney injury (AKI) caused by sepsis is a serious disease which mitochondrial oxidative stress and inflammatory play a key role in its pathophysiology. Ceria nanoparticles hold strong and recyclable reactive oxygen species (ROS)-scavenging activity, have been applied to treat ROS-related diseases. However, ceria nanoparticles can't selectively target mitochondria and the ultra-small ceria nanoparticles are easily agglomerated. To overcome these shortcomings and improve therapeutic efficiency, we designed an ROS-responsive nano-drug delivery system combining mitochondria-targeting ceria nanoparticles with atorvastatin for acute kidney injury. Methods: Ceria nanoparticles were modified with triphenylphosphine (TCeria NPs), followed by coating with ROS-responsive organic polymer (mPEG-TK-PLGA) and loaded atorvastatin (Atv/PTP-TCeria NPs). The physicochemical properties, in vitro drug release profiles, mitochondria-targeting ability, in vitro antioxidant, anti-apoptotic activity and in vivo treatment efficacy of Atv/PTP-TCeria NPs were examined. Results: Atv/PTP-TCeria NPs could accumulate in kidneys and hold a great ability to ROS-responsively release drug and TCeria NPs could target mitochondria to eliminate excessive ROS. In vitro study suggested Atv/PTP-TCeria NPs exhibited superior antioxidant and anti-apoptotic activity. In vivo study showed that Atv/PTP-TCeria NPs effectively decreased oxidative stress and inflammatory, could protect the mitochondrial structure, reduced apoptosis of tubular cell and tubular necrosis in the sepsis-induced AKI mice model. Conclusions: This ROS-responsive nano-drug delivery system combining mitochondria-targeting ceria nanoparticles with atorvastatin has favorable potentials in the sepsis-induced AKI therapy.

Keywords: ROS-responsive; acute kidney injury; ceria; mitochondria-targeting.; oxidative stress.

Scheme 1

Schematic illustration of Atv/PTP-TCeria NPs for acute kidney injury. Ceria NPs are modified with triphenylphosphine (TCeria NPs) to be endowed the ability to target mitochondria, then TCeria NPs are coated by mPEG-TK-PLGA polymer and load atorvastatin. Atv/PTP-TCeria NPs could passively target the kidney and release drug responding to high-level ROS and TCeria NPs would target mitochondria to scavenge excessive ROS for ameliorating AKI.

Figure 1

Synthesis and characterization of TCeria NPs. (A) Size distributions and TEM images (scale bar = 20 nm) of Ceria NPs and (B) TCeria NPs. (C) XPS spectrum of TCeria NPs. (D) XPS analysis of Ce 3d, Peaks at 881 and 887 eV are related to Ce ⁴⁺ and Ce ³⁺, peak at 915 eV indicates the presence of Ce ⁴⁺. (E) FT-IR spectrums of TPP, Ceria NPs, and TCeria NPs.

Figure 2

Synthesis and characterization of Atv/PTP-TCeria NPs. (A) ¹H-NMR spectra of mPEG, PLGA, and mPEG-TK-PLGA from top to bottom, respectively. (B) Hydrodynamic diameter distributions and zeta potentials of TCeria, PTP-TCeria, Atv/PTP-TCeria NPs and TEM images of PTP-TCeria and Atv/PTP-TCeria NPs dispersed in DI water, scale bar = 20 nm. (C) Diameter changes of PTP-TCeria NPs cultivated with 100 mM H₂O₂. (D) Drug release behaviors of Atv/PTP-TCeria NPs. The release of atorvastatin was observed with time in the presence of 0 μM/100 μM/ 200 μM H₂O₂ in PBS buffer at pH 7.4.

Figure 3

Cellular uptake and mitochondria-targeting ability of TCeria NPs. (A) Images of HUVECs treated with FITC-Ceria NPs, FITC-TCeria NPs at a dose of 0.100 mM for 20 min, 40 min,1 h and 2 h with mitochondria stained, scale bar = 50 μm. (B) Images of HUVECs treated with FITC-Ceria NPs, FITC-TCeria NPs for 2 h with mitochondria stained, scale bar = 10 μm. (C) The colocalization analysis of the fluorescent intensity of Ceria NPs, TCeria NPs and mitochondria in cells from figure 3B.

Figure 4

In vitro antioxidant capacity of Atv/PTP-TCeria NPs. (A) Cell viability of TCeria NPs, (B) PTP-TCeria NPs in HUVECs after 24 h exposure. (C) Cell viability of H₂O₂-stimulated HUVECs after incubated with atorvastatin, PTP-TCeria, PTP or Atv/PTP-TCeria NPs for 24 h. (D) Semi-quantitative results of intracellular ROS in figure 4g. (E) SOD activity and (F) MDA level of H₂O₂-stimulated HUVECs after incubated with atorvastatin, PTP-TCeria, PTP or Atv/PTP-TCeria NPs for 24 h. (G) Intracellular ROS of H₂O₂-stimulated HUVECs after incubated with atorvastatin, PTP-TCeria, PTP or Atv/PTP-TCeria NPs for 24 h was observed via fluorescent microscopy, scale bar = 100 μm. Data are expressed as the mean ± SD, n = 5 for each group.

Figure 5

In vitro anti-apoptotic activity of Atv/PTP-TCeria NPs. H₂O₂-stimulated HUVECs were incubated with atorvastatin, PTP-TCeria, PTP and Atv/PTP-TCeria NPs for 24 h. (A) The Apoptosis in different groups was evaluated by flow cytometry. (B) The level of Caspase-3 in different groups was evaluated by western blot. (C) The semi-quantitative analysis of Caspase-3. (D) The Δψm of mitochondria in different groups was investigated using confocal microscopy, scale bar = 20 μm. (E) Semi-quantitative results of flow cytometry (F) Semi-quantitative results of the Δψm changes of mitochondria. Data are expressed as the mean ± SD, n = 3 for each group.

Figure 6

In vivo biodistribution and anti-inflammatory, antioxidant activity of Atv/PTP-TCeria NPs. (A) AKI mice were injected intravenously with ICG loading PTP-TCeria (1 mg/kg) and visualized at 6h after administration. (B) The kidney fluorescence intensity in ICG loading PTP-TCeria NPs and free ICG treated AKI mice model at different times. (C) The semi-quantitation of fluorescence intensity in figure 6a. (D) The semi-quantitation of fluorescence intensity in figure 6b. (E) The levels of TNF-α, (F) IL-6, (G) SOD, (H) MDA in the kidney at 24 h and 48 h after different treatments.

Figure 7

Histopathological changes, assessment on kidney function after Atv/PTP-TCeria NPs therapy and anti-apoptotic efficacy of Atv/PTP-TCeria NPs. (A) Hematoxylin and eosin (H&E)-stained kidney sections of the AKI mice after different treatments (atorvastatin, PTP-TCeria NPs, PTP and Atv/PTP-TCeria NPs) (at 48 h), Scale bar = 100 μm. Apoptosis cells staining using TUNEL assay (brown nuclei) of different groups (at 48 h), Scale bar = 50 μm. (b and c) Scrum and BUN levels at 24 and 48 h in the AKI mice after different treatments. (D) Cell necrosis scores. (E) The quantitation of TUNEL-positive cells.

Figure 8

In vivo protective effect on mitochondrial structure of Atv/PTP-TCeria NPs. TEM images of mitochondria from representative samples in the control, AKI, and Atv/PTP-TCeria NPs treated groups (at 24 h and 48 h). Scale bar = 1 μm. N means nucleus.

Figure 9

The evaluation of the adverse effects of different treatments. (A) Histopathology images of heart, liver, spleen, lung, and kidney collected from the control, AKI and Atv/PTP-TCeria NPs treated groups (at 48 h), scale bar = 100 μm. Concentrations of (B) ALT, (C) AST in the AKI mice after different treatments (atorvastatin, PTP-TCeria NPs, PTP and Atv/PTP-TCeria NPs). *p<0.05 compared with the AKI group.