Mitochondrial Targeted Cerium Oxide Nanoclusters for Radiation Protection and Promoting Hematopoiesis

Abstract

Purpose: Mitochondrial oxidative stress is an important factor in cell apoptosis. Cerium oxide nanomaterials show great potential for scavenging free radicals and simulating superoxide dismutase (SOD) and catalase (CAT) activities. To solve the problem of poor targeting of cerium oxide nanomaterials, we designed albumin-cerium oxide nanoclusters (TPP-PCNLs) that target the modification of mitochondria with triphenyl phosphate (TPP). TPP-PCNLs are expected to simulate the activity of superoxide dismutase, continuously remove reactive oxygen species, and play a lasting role in radiation protection.

Methods: First, cerium dioxide nanoclusters (CNLs), polyethylene glycol cerium dioxide nanoclusters (PCNLs), and TPP-PCNLs were characterized in terms of their morphology and size, ultraviolet spectrum, dispersion stability and cellular uptake, and colocalization Subsequently, the anti-radiation effects of TPP-PCNLs were investigated using in vitro and in vivo experiments including cell viability, apoptosis, comet assays, histopathology, and dose reduction factor (DRF).

Results: TPP-PCNLs exhibited good stability and biocompatibility. In vitro experiments indicated that TPP-PCNLs could not only target mitochondria excellently but also regulate reactive oxygen species (ROS)levels in whole cells. More importantly, TPP-PCNLs improved the integrity and functionality of mitochondria in irradiated L-02 cells, thereby indirectly eliminating the continuous damage to nuclear DNA caused by mitochondrial oxidative stress. TPP-PCNLs are mainly targeted to the liver, spleen, and other extramedullary hematopoietic organs with a radiation dose reduction factor of 1.30. In vivo experiments showed that TPP-PCNLs effectively improved the survival rate, weight change, hematopoietic function of irradiated animals. Western blot experiments have confirmed that TPP-PCNLs play a role in radiation protection by regulating the mitochondrial apoptotic pathway.

Conclusion: TPP-PCNLs play a radiologically protective role by targeting extramedullary hematopoietic organ-liver cells and mitochondria to continuously clear ROS.

Keywords: ceria nanoclusters; mitochondria targeting; oxidative stress; radioprotective effect; superoxide dismutase.

Scheme 1

Schematic diagram based on mitochondria targeted biocompatibility ceria nanoclusters used for radioprotection and promoting hematopoiesis.

Figure 1

Synthesis and characterization of CNLs, PCNLs and TPP-PCNLs. (A) Schematic diagram of the synthesis process of TPP-PCNLs. (B–E) HR-TEM, negative staining of tungsten phosphide for PCNLs (B and C) and TPP-PCNLs (D and E). (F) Hydrated particle size analysis of CNLs, PCNLs and TPP-PCNLs. (G) Stability analysis and characterization of TPP-PCNLs by DLS.

Figure 2

Characterization of CNLs, PCNLs and TPP-PCNLs. (A) UV mapping of TPP-PCNLs. (B) FTIR spectrum of the CNLs, PCNLs and TPP-PCNLs. (C) X-ray Photoelectron Spectroscopy (XPS) of TPP-PCNLs. (D) Analysis of SOD activity of CNLs, PCNLs and TPP-PCNLs; (E) Analysis of CAT activity. (F) Confocal microscopy analysis of PCNLs and TPP-PCNLs and cellular localization analysis with Cy3 amine labeling (red) and Mito-Tracker (green).

Figure 3

TPP can improve mitochondrial morphology and function damage caused by radiation. (A) Effects of PCNLs and TPP-PCNLs on L-02 cell viability. (B) Effect of TPP-PCNLs on the survival rate of L-02 cells after 6Gy irradiation. (C) ATP energy levels of L-02 cells. (D) Transmission electron microscopy (TEM) images of L-02 cells. Mitochondria vacuolization is indicated by an green arrow. (E) Fluorescence image and quantitative analysis of the MMP of L-02 cells (100×) detected by the JC-1 method. (F) Fluorescence image and quantitative analysis of the Super oxide of L-02 cells (100×) detected by the Mito-SOX method. *Represents a significant difference compared with the IR group, *P<0.05, **P<0.01; ^#Represents an extremely significant difference compared between PCNLs and TPP-PCNLs, ^#P<0.05, ^##P<0.01.

Figure 4

Radioprotection effect of TPP-PCNLs in irradiated cells. Images and quantitative analysis of L-02 cells treated with PCNLs and TPP-PCNLs. (A) Fluorescence image of the ROS of L-02 cells (100×) detected by the DCFH-DA method. (B) Image of cell colony formation. L-02 cells were pretreated with TPP-PCNLs, and colony formation was examined 10 days after 6Gy irradiation (40×). *Represents a significant difference compared with the IR group, *P<0.05, **P<0.01; ^#Represents an extremely significant difference compared between PCNLs and TPP-PCNLs, ^#P<0.05, ^##P<0.01.

Figure 5

Cellular mechanism of radiation protection of TPP-PCNLs. (A) Comet image of DNA damage in L-02 cells (100×). (B) Detection and quantitative analysis of apoptosis-related protein expression in irradiated L-02 cells treated with TPP-PCNLs. *P<0.05 vs IR (+), **P<0.01 vs IR (+).

Figure 6

Radioprotection effect of TPP-PCNLs in vivo. (A) Changes in ceria content in the blood of mice at different time points after CNLs, PCNLs and TPP-PCNLs treatment. (B) Changes in ceria content in the heart, liver, spleen, lungs and kidneys of mice after CNLs, PCNLs and TPP-PCNLs treatment. (C–G) Assessment of radioprotective effect of TPP-PCNLs on WBI mice. (H) Weight of the mice in each group. (I and J) Splenic nodules. The nucleated cells in bone marrow were counted. (K–M) Changes in red blood cell counts (RBCs), white blood cell counts (WBCs) and platelets (PLTs) in the peripheral blood of mice at different time points. (N–P) Levels of glutathione peroxidase (GP-x), malondialdehyde (MDA) and glutathione (GSH) in the serum of the mice in each group. *Represents a significant difference compared with the IR group, *P<0.05, **P<0.01.

Figure 7

Histopathological changes in the bone marrow (A), small intestine (B), liver (C), spleen (D), kidney (E) and heart (F) in each group were evaluated by hematoxylin and eosin (H&E) staining.