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Highly Catalytic Nanodots with Renal Clearance for Radiation Protection

Ionizing radiation (gamma and x-ray) is widely used in industry and medicine, but it can also pose a significant hazardous effect on health and induce cancer, physical deformity and even death, due to DNA damages and invasion of free radicals. There is therefore an urgent unmet demand in designing highly efficient radioprotectants with synergetic integration of effective renal clearance and low toxicity. In this study, we designed ultrasmall (sub-5 nm) highly catalytically active and cysteine-protected MoS2 dots as radioprotectants and investigated their application in protection against ionizing radiation. In vivo preclinical studies showed that the surviving fraction of MoS2-treated mice can appreciably increase to up to 79 % when they were exposed to high-energy ionizing radiation. Furthermore, MoS2 dots can contribute in cleaning up the accumulated free radicals within the body, repairing DNA damages and recovering all vital chemical and biochemical indicators, suggesting their unique role as free radical scavengers. MoS2 dots showed rapid and efficient urinary excretion with more than 80 % injected dose (I.D.) eliminated from the body after 24 hours due to their ultrasmall hydrodynamic size and did not cause any noticeable toxic responses up to 30 days.


Introduction
High-energy ionizing radiations (X-rays and gamma rays) are widely used in industry and medicine, but these radiations also cause significant health hazards such as cancer and other related diseases. [1][2][3][4][5][6] In clinical medicine, more than 50% cancer patients need to receive radiation therapies, but high-energy radiations during the treatments not only kill cancer cells but also cause inevitable damages to normal tissues. [7][8] Besides, more and more nuclear power plants are proposed in the world due to the rising demands in energy, imposing potential radiation risks on public health. [9][10] Under exposures to ionizing radiations, lots of free radicals including reactive oxygen species (ROS) are formed through ionizing reactions such as the photoelectric, Compton scattering and Auger effects. [11][12] These free radicals are able to react with DNA and RNA inside the body, cause structural and functional changes and affect biological processes. [13][14][15] As a result, it induces cell apoptosis and further triggers cancer or even death. The use of radioprotectants provides a feasible solution to shield heath tissues from high-energy radiations. 16 Amifostine (Ethyol ® ) is an extensively used prescription radioprotectant in radiation medicine by scavenging oxygen-derived free radicals, but its blood elimination half-life is only 1 minute, limiting its scavenging activities against ROS. 16 Other materials have shown abilities of radiation protection to an extent, but they cannot afford effective excretions which could result in potential hepatic and splenic toxicities. [14][15] As a matter of fact, US Food and Drug Administration (FDA) have required all injected agents for in vivo uses to be completely cleared from the body. 17 Therefore, it is highly desirable to explore an ideal radioprotectant with capabilities of highly efficient removals of ROS, renal clearance and low toxicities, for clinical translation as an adjuvant in radiotherapies.
To address these critical challenges, we utilized a simple approach towards the synthesis of ultrasmall cysteine-protected MoS2 dots as radioprotectants. Surface protection with zwitterionic cysteine offers several indispensable merits to the MoS2 dots. Firstly it avoids the significant increase in hydrodynamic sizes as compared to other surface ligands and thus allows effective elimination from renal clearance. [18][19] Secondly, the aqueous dispersibility and stability are significantly enhanced by the surface modification, maintaining the ultrasmall size in vivo and refraining from aggregation. Last, non-specific adsorption of serum proteins, especially opsonin, is largely prohibited, leading to postponed removal from the body and a relatively longer circulation time in blood to achieve desired radiation protection. The as-prepared MoS2 dots exhibited extraordinary electrocatalytic activities for hydrogen peroxide and oxygen reduction reactions (ORRs), leading to lots of free electron transfers. Its endogenous catalytic properties provide a promising and effective pathway for scavenging free radicals in vivo via rapid reactions with oxygen radical superoxide (O 2-) and non-radical oxidant hydrogen peroxide (H2O2) in blood.
Owing to their strong catalytic activities, MoS2 dots improved the surviving fraction of mice exposed to high radiation doses of 662 keV gamma ray. Furthermore, the ultrasmall MoS2 dots can indeed eliminate the ROS through reduction reactions in major organs. As a result, superoxide dismutase (SOD), as an important indicator for antioxidant defense in almost all living cells under exposure to oxygen, nearly recovered back to normal levels, suggesting the repair of radiation-induced damages. The ultrasmall cysteine-protected MoS2 dots can be rapidly excreted via kidney and did not cause any toxicological responses in 30 days post injection.

Results and Discussion
Ultrasmall MoS2 dots were synthesized and purified by a combinational approach of ultrasonication and gradient centrifugation according to our published procedures. [20][21][22] However, it is difficult to disperse bare MoS2 dots as a stable colloid solution in water, preventing their further applications in biological systems. A cysteine protection layer with a molecular weight of 121 Da was introduced which not only maintains the ultrasmall hydrodynamic size to meet the cut-off size of <5.5 nm for renal clearance, 17 but also endows excellent biocompatibility in biomedical applications. Cysteine-protected MoS2 dots were obtained and stabilized through the chemical bonding and van der Waals interactions between MoS2 and cysteine, 23 and were further purified and re-dispersed in water (Figure 1a). Obvious Tyndall effects were observed, indicative of the existence of small particles in solution. Figure 1b (Figure 1c). Additionally, the S 2p3/2 binding energy located at 164.0 eV suggested the formation of disulfide bonds which are ascribed to the conjugation of cysteine and MoS2 dots (Figure 1d). 24 On the other hand, no signals from disulfide bonds can be found on the corresponding counterparts, cysteine and unprotected MoS2 dots ( Figure S2). Optical absorption was investigated by UV-vis spectroscopy (Figure 1e). Cysteine alone did not show any absorption features, while the MoS2 suspension and cysteine-protected MoS2 dots displayed multiple absorption peaks at 393, 470, 607 and 670 nm, attributed to deep energy levels of electronic transitions and interband excitonic transitions. [25][26] Notably, the results are similar to the absorption bands observed for MoS2 particles prepared by other methods. [25][26] We evaluated the in vitro catalytic activities of cysteine-protected MoS2 dots towards reduction of H2O2 in N2-saturated 0.01 M phosphate-buffered salin (PBS), pH = 7.4, using a cysteine-MoS2 modified glassy carbon electrode (GCE). Figure 1f showed the cyclic voltammetric (CV) curves of the ultrasmall cysteine-protected MoS2 dots in the presence and absence of 5.00 mM H2O2 at the scan rate of 50 mV·s −1 . Negligible reduction current was observed in the absence of H2O2, whereas in the presence of H2O2, the reduction current increased sharply when the scan potential shifted to more negative than -0.4 V, indicating extraordinary catalytic activities toward reduction of H2O2 compared to voltammetric responses of the bare GCE without modification of cysteine-protected MoS2 dots ( Figure S3a) We firstly examined the in vitro toxicities of cysteine-protected MoS2 dots in 3T3/A31 cells using neutral red assay and MTT method which showed extremely low cytotoxicities with doses up to 145 μg/ml ( Figure S4). Viabilities of cells treated with cysteine-protected MoS2 dots in different doses under exposures to gamma ray were significantly increased compared with those without treatments (Figure 2a), suggesting the powerful in vitro protective behaviors of cysteineprotected MoS2 against radiation. The DNA damages of cysteine-protected MoS2 dots with different concentration up to 145 μg/ml also investigated by single-cell sol-gel electrophoresis, and no significant difference on tail moments was found, suggesting negligible DNA damages from MoS2 dots ( Figure S5). DNA damages arising from radiation were further estimated ( Figure S6). No obvious cell tail moments were found in the control, but cell tails implying DNA damages were easily identifiable with exposures to high-energy gamma ray. In contrast, the cell tails from cells treated with cysteine-protected MoS2 dots showed distinct recoveries, presenting effective DNA repairs. Quantitative investigations of tail moments showed that cells treated with cysteine-protected MoS2 dots recovered to an appreciable degree from exposures of gamma ray, as compared to cells treated only with radiation (Figure 2b).
In vivo protection from radiation using cysteine-protected MoS2 dots were investigated with C57BL/6 mice. 200 μL cysteine-protected MoS2 dots at different concentrations were intraperitoneally injected into mice. The mice were then exposed to high energy gamma ray ( 137 Cs, 3600 Ci) at the dose of 7.5 Gy. The surviving fractions of radiation-injured mice with various injection concentrations are shown in Figure 2c. The surviving fraction of mice exposed to high-energy radiations without injection of cysteine-protected MoS2 dots is 0 after two weeks.
This result demonstrated radiation induced acute damages and death which are consistent with observations in previous published work. 6 With injection of cysteine-protected MoS2 dots, the surviving fractions of mice were 7.1 and 42.9 and 78.6 % at the doses of 10, 20 and 50 mg/kg. Meanwhile, the surviving fraction of mice treated with cysteine-protected MoS2 dots (200 μL, 5 mg/ml) in the absence of radiation is 100 %, indicating low toxicities at this concentration. In fact, cysteine protection layers were observed to show outstanding biocompatibility, in good agreement with previous results. [27][28] Besides, we also investigated the in vivo radiation protection effects of Pt-, Co-and Ni-doped MoS2 dots as well as cysteine, but none of these metal-doped MoS2 dots showed comparable survival rates to that of cysteine-protected MoS2 dots (Figure S7), which may be due to the large sizes and different surface chemistries. Noticeably, a low survival rate was observed for cysteine-treated mice, clearly providing strong evidences that the radiation protection effect against gamma ray is from MoS2 instead of cysteine. Next, DNA damages of irradiated mice treated with or without cysteine-protected MoS2 were assessed (Figure 2d). Total DNA from bone marrow cells and bone marrow nucleated cells (BMNC), two dominant indicators of ionizing radiation, were collected from control mice, irradiated mice and irradiated mice treated with cysteine-protected MoS2. DNA from healthy mice showed an optical density (OD) of 0.42 after 1 day, but it drastically decreased to 0.22 only after 1 day post exposure to radiation, indicating severe DNA damages induced by radiation. In contrast, OD of total DNA for mice treated with cysteine-protected MoS2 dots decreased to a higher value of 0.29 after 1 day.
After 7 days, OD of mice treated with cysteine-protected MoS2 dots rose to 0.38, almost recovered to the healthy value, compared to that of 0.25 from mice with only radiation. These results illustrate that cysteine-protected MoS2 dots can effectively decrease DNA damages from high-energy radiations. The number of BMNC is presented as in Figure S8. Similar to the results of DNA damages, the BMNC number decreased from 6×10 6 cells/mL in healthy mice to 4.3×10 6 cells/mL in irradiated mice after 1 day, while it was recovered to 4.6×10 6 cells/mL in irradiated mice treated with cysteine-protected MoS2 dots. On the 7 th day, the BMNC number in mice treated with MoS2 dots was stably maintained at 4.6×10 6 /mL as to a significantly decreased level of 1.3×10 6 /mL in mice only treated with radiation. The results clearly presented that cysteineprotected MoS2 dots are critical in recovering the number of BMNC.
To address long-term damages caused by radiation, blood chemistry panels of irradiated mice under 7 Gy gamma ray were studied ( Figure S9). In the beginning (1 day), it is clear that white blood cells (WBC), red blood cells (RBC) and platelets (PLT) sharply decreased after exposure to gamma ray indicating strong radiation-induced inflammatory responses, independent of treatments of cysteine-protected MoS2 dots. At the middle time point (7 days), WBC and PLT were still in low levels without distinct recoveries to healthy levels, while all other indicators started to display certain recoveries after treatments with cysteine-protected MoS2 dots. After 30 days, a large number of the indicators of mice treated with irradiation showed significant decreases, but those receiving treatments of cysteine-protected MoS2 dots have appreciably recovered to normal levels. Besides, radiation triggered significant changes in levels of alanine aminotransferase (ALT) and serum creatinine (CERA) 1 and 7 days post injection ( Figure S10).
On the other hand, all these indicators for MoS2-dot-treated mice were thoroughly restored to normal 30 days post injection, while irradiated mice without MoS2 treatments presented significant variations in the levels of ALT, albumin (ALB), blood urine nitrogen (BUN) and globulin (GLOB). Under exposure to radiations, the acute radiations not only give rise to severe DNA breaks and considerable decrease in the BMNC numbers, but also instantaneously elicit inflammatory responses. 29 Radiation-induced damages were the most severe after 7 days and lots of irradiated mice even began to die. After that, the mice started to recover gradually up to the 30 th day. Undoubtedly, radiation always induces certain irreparable damages of DNA and BMNC and thus strongly affects the panels of blood chemistry and biochemistry, implying infections and inflammations. However, the cysteine-protected MoS2 dots can free the mice from radiationrelated DNA and BMNC damages, relieve corresponding infections and thus increase the overall surviving fractions of mice.
To further reveal the mechanism of the underlying radiation protection, superoxide dismutase (SOD) and 3,4-Methylenedioxyamphetamine (MDA) were employed to provide some insights. SOD are enzymes that alternately catalyze dismutation (or partitioning) of the superoxide (O2 − ) radicals into either ordinary molecular oxygen (O2) or hydrogen peroxide (H2O2). In contrast, MDA is a harmful product generated by mice. The contents of SOD and MDA indicate levels of damages caused by ionizing radiation. Figure 3a and b showed SOD levels in lung and liver. When mice were exposed to gamma ray, the SOD of lung from all mice, independent of treatments of MoS2 dots, notably decreased after 1 day. However, 7 days post radiation, the SOD from the mice treated with cysteine-protected MoS2 dots exhibited powerful recoveries compared to mice without treatments, clearly elucidating the increase in the ability of ROS clearance. Figure 3c and d examined the MDA levels of lung and liver from irradiated mice with or without treatments of cysteine-protected MoS2 dots. The MDA level in the lung of normal mice was 8.1 nmol/mL, but it sharply increased to 22.3 nmol/mL after 1 day (Figure 3c).
In the meantime, the treatment with cysteine-protected MoS2 dots could partially shield the mice from radiation damages with much lower MDA levels of 15.1 nmol/mL after 1 day. After 7 days, the MDA levels decreased to 10 nmol/mL for mice with treatments of cysteine-protected MoS2 dots compared to 12.8 nmol/mL for mice only with radiation. Similarly, Figure 3d indicated the MDA levels in the liver sharply increased from 3.6 nmol/mL to 13.2 and 12.1 nmol/mL for treated and untreated mice, respectively. After 7 days, the mice treated with cysteine-protected MoS2 dots recovered back to 4 nmol/mL, but untreated mice was still 5.5 nmol/mL, significant higher than the threshold value of normal mice without irradiations. In healthy mice, SOD is maintained at normal levels, 30-31 but high-energy irradiation is well known to cause generation of excessive amount of ROS and consumption of large quantities of SOD. Consequently, the SOD levels from liver and lung decrease enormously. The cysteine-protected MoS2 dots can help to get rid of the undesirable hazardous ROS in vivo and rescue the body from consuming lots of SOD to achieve the removal of ROS and accordingly, SOD can be recovered and reserved at the normal level. Similarly, MDA is normally present in low levels in the body. When mice suffered from the exposure to radiation, a large quantity of MDA is produced due to the generation of ROS in excess. The cysteine-protected MoS2 dots can decrease MDA levels of mice via eliminating free radicals. As such, as the cysteine-protected MoS2 dots with reducing potencies circulate into blood, they reduce the radiation-induced ROS, functioning as free radical scavengers.
For further medical applications, pharmacokinetics and biosafety are the two most important characteristics to be taken into consideration. We investigated the pharmacokinetics, urine excretion and in vivo toxicities of cysteine-protected MoS2 dots at the injection dose of 50 mg/kg. The C57/BL6 mice were injected at the concentration of 5 mg/ml with blood and urine collected for pharmacokinetic and renal-excretion studies, as measured by inductively coupled plasma mass spectrometry (ICP-MS). In addition, we performed the standard biochemistry examination on the mice treated with cysteine-protected MoS2 dots at different time points of 1, 7 and 30 days (Figure 4e and Figure   S12b). The biochemical parameters including ALT, AST, total protein (TP), ALB, blood urea nitrogen, CREA, GLOB, and total bilirubin (TBIL) were investigated. We emphasize ALT, AST, and CREA, because they are closely related to the functions of liver and kidney of mice. ALT showed increase after 7 days, but recovered to normal after 30 days treatments. AST and CREA did not have statistical differences in the treated mice. In fact, MoS2 nanosheets and other twodimensional nanomaterials have shown potential applications in imaging and photothermal therapies, but the hydrodynamic sizes of these particles are too much larger than the 5.5 nm cutoff value of kidney, which will induce potential liver toxicities. [36][37][38][39][40][41][42][43] For example, Au nanoparticles coated by PEG and BSA caused acute damages even after 30 days at a relatively low injection dose of 5 mg/kg, [44][45] In comparison, the sub-5 nm MoS2 dots we prepared with efficient renal clearance will escape from the uptake of the reticuloendothelial system (ROS), minimizing the toxic effects. The cysteine-protected MoS2 dots showed extremely low liver toxicities even at a 10-fold high dose of 50 mg/kg. Finally, the pathological changes of organs were demonstrated by immunohistochemistry at different time points. Liver, spleen, kidney and other organs were collected and sliced for Haematoxylin and Eosin (H&E) staining (Figure 4f and Figure S13). No apparent damages were observed in all organs, especially the spleen and kidney during the entire period.
Radiation protection is utmost important in both healthcare and environment. Fukushima nuclear radiation has caused detrimental public crisis and irreversible radiation damages in large areas. 46 Thus, the development of radioprotectants with low toxicities or even without toxicities is promising for potential applications in this area. Cysteine-protected MoS2 dots not only endow renal clearance, but also offer strong catalytic properties that allow to react with excessive radiation-induced ROS in the blood of mice. However, we have conceived that it still has large room for the development of new materials for protection from ionizing radiation. In the future, it is valuable to explore new small-molecular organic materials with high activities in free radical scavenging. It is necessary to develop effective radioprotectants with sub-5 nm hydrodynamic sizes as well as low toxicities. [47][48][49][50][51][52][53][54] Besides, it is of great importance to design materials with well-defined functional surfaces for radiation protection. It is also interesting to combine features of energy transfers and energy-dependent responses under exposure to high-energy radiation.
Detailed biological and bio-catalytic mechanisms of radiation protection are still necessary to be further investigated. [55][56][57]

Conclusion
In summary, we developed ultrasmall cysteine-protected MoS2 dots radioprotectants, with high efficiencies in renal clearance and radiation protection against gamma ray. This type of novel nanomaterials behave like organic molecules with low levels of retentions in RES organs such as liver and spleen, avoiding associated toxicities. The cysteine-protected MoS2 dots also 4x10 3 A31 cells were seeded into five 96-well plates, supplemented with high-glucose DMEM containing 10% FBS. After 10 hours of incubation, the cells were treated with 0.2 μg and 2 μg MoS2 dots for 1 hour which were dissolved in 100 ul complete culture media. The five plates were then exposed to 0, 2, 4, 8 and 10 Gy radiations respectively. After 48 hours, NR was used to evaluate cell viabilities. When cells had sufficiently taken up NR in about~3 hours, the media containing NR was discarded and each well was gently washed with 1X PBS, followed by addition of 150 μl desorb solution to extract NR from living cells. All the plates were homogenized on a microtiter plate shaker for 5 min and the optical absorbance was recorded at 540 nm by Infinite F200 multimode plate reader.
In Vitro DNA Damages. The in vitro protocols were approved by the Institutional Review

Board of Institute of Radiation Medicine at the Chinese Academy of Medical Sciences (CAMS).
A modified protocol of alkaline COMET assay was used to evaluate DNA damages. Agarose (0.8%, 500 μl) was paved homogeneously on glass microscopic slides. After solidification, 4.8 x10 4 cells in 30 μL 1X PBS incubated with MoS2 dots (0.5-135 μg/ml) were mixed with 70 μL low-melting-point agarose (0.6%) and 20 μl of this mixture was spread over the slide completely.
The solidified slides were placed into freshly-prepared ice-cold lysis buffer (2.5 mol/L NaCl, 100 mmol/L Na2EDTA, 10 mmol/L Tris-HCl, 10% DMSO, 1% Triton X-100) for 2 hours, followed by immersing them in a horizontal gel electrophoresis unit which was filled with chilled electrophoresis buffer (1 mmol/L Na2EDTA, 300 mmol/L NaOH, pH 7.4 for 30 min. homogenates were employed. Equal volumes (100 μl) of organ sample, ethanol and tetrathoxypropane (10 nmol/ml) were added into individual 5 ml EP tubes, among which ethanol and tetrathoxypropane were negative and positive controls respectively. 100 ul tissue lysis solution was added into each 5ml EP tube and mixed completely. Trichloroacetic acid (10 %, 3 ml) and thiobarbituric acid (0.6 %, 1 ml) were added into the system orderly. Then the tubes were heated in water at 95℃ for 40 minutes. After being cooled down to room temperature, all the samples were centrifuged at 3500~4000 r/min for 10 minutes with the supernatants analyzed by UV-vis spectrophotometer at 532 nm (Shimadzu, UV-1750). were sacrificed accordingly at each time point using isoflurane anesthetic and angiocatheter exsanguination with 1X PBS. Blood and organs were collected for biochemistry and pathological analyses. One mouse from each group was fixed with 10% formalin in PBS following exsanguination with PBS. During necropsy, liver, kidneys, spleen, heart, lung, testis, brain, bladder and thymus were collected and weighed.

In Vivo
Hematology, Biochemistry and Pathology: Using a standard saphenous technique, vein blood was collected in potassium EDTA collection tubes for hematological analysis. Standard hematological and biochemical examinations were performed. 1 mL of blood was collected from mice and separated into cellular and plasma fractions by centrifugation. Mice were sacrificed by isoflurane anesthetic and angio catheter exsanguinations. Major organs were harvested, fixed in 10% neutral buffered formalin, processed routinely into paraffin and stained by H&E, with pathology examined using a digital microscope.
Statistical Analyses: Paired Student's t-test was used for statistical analysis between the control group and the treatment group (MoS2 alone or radiation+MoS2). The Analysis of Variance (ANOVA) was used for statistical analysis among the control, radiation, and radiation+MoS2 groups. Differences were considered statistically significant at a P value < 0.05.

Figures and Figure Captions
Schematic illustration of radiation protection with cysteine-protected MoS2 dots