Highly Catalytic Niobium Carbide (MXene) Promotes Hematopoietic

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Highly Catalytic Niobium Carbide (MXene) Promotes Hematopoietic Recovery after Radiation by Free Radical Scavenging Xiangyi Ren, Minfeng Huo, Mengmeng Wang, Han Lin, Xuxia Zhang, Jun Yin, Yu Chen, and Honghong Chen ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b09327 • Publication Date (Web): 03 Jun 2019 Downloaded from http://pubs.acs.org on June 5, 2019

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Highly Catalytic Niobium Carbide (MXene) Promotes Hematopoietic Recovery after Radiation by Free Radical Scavenging

Xiangyi Ren1, Minfeng Huo2,3, Mengmeng Wang1, Han Lin2,3, Xuxia Zhang1, Jun Yin1, Yu Chen2* and Honghong Chen1*

1Department

of Radiation Biology, Institute of Radiation Medicine, Fudan University, Shanghai, 200032, P. R. China.

2State

Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai

Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, P. R. China. 3University

of Chinese Academy of Science, Beijing, 100049, P. R. China

E-mail: [email protected]; [email protected]

1

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ABSTRACT：Ionizing radiation (IR) has been extensively used in industry and radiotherapy, but IR exposure from nuclear or radiological accidents often causes serious health effects in an exposed individual, and its application in radiotherapy inevitably brings undesirable damages to normal tissues. In this work, we have developed ultrathin two-dimensional (2D) niobium carbide (Nb2C) MXene as a radioprotectant and explored its application in scavenging free radicals against IR. The 2D Nb2C MXene is featured with the intriguing antioxidant properties in effectively eliminating hydrogen peroxide (H2O2), hydroxyl radicals (•OH) and superoxide radicals (O2•-). The pre-treatment with biocompatible polyvinyl pyrrolidone (PVP)-functionalized Nb2C nanosheets (Nb2C-PVP NSs) significantly reduces IR-induced production of reactive oxygen species (ROS), resulting in enhanced cell viability in vitro. A single intravenous injection of Nb2C-PVP significantly enhances the survival rate of 5 Gy and 6.5 Gy irradiated mice to 100% and 81.25%, respectively, and significantly increases the bone marrow mononuclear cells (BM-MNCs) after IR. Critically, Nb2C-PVP reverses the damages of the hematopoietic system in irradiated mice. Single administration of Nb2C-PVP significantly increases superoxide dismutases (SODs) activities, decreases malondialdehyde (MDA) levels, and thereby reduces IR-induced pathological damage in the testis, small intestine, lung, and liver of 5 Gy irradiated mice. Importantly, Nb2C-PVP is almost completely eliminated from the mouse body on day 14 post-treatment and no obvious toxicities are observed during the 30-day post-treatment period. Our study pioneers the application of 2D MXenes with intrinsic radioprotective nature in vivo.

KEYWORDS: Nb2C, MXenes, radiation protection, free radical scavenger, superoxide dismutases, hematopoiesis 2


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With the broad application of nuclear technology in the fields of industry, agriculture and medicine, there is an increasing risk of human radiation exposure. Ionizing radiation (IR)-induced health hazards such as acute radiation syndrome and cancer have caused worldwide attention.1-3 While radiotherapy remains the mainstay of treatment for malignant tumors, the radiation also brings severe side effects in patients.4,5 Therefore, it is an urgent demand to develop potent radioprotectants for clinical uses. Over the years, researchers are dedicated to developing effective radioprotectors via various strategies including free radical scavenging, anti-oxidation, immune stimulation, and toll-like receptor agonist.6-10 However, very few radioprotectors have been developed for clinical uses, and their radio-protective efficiencies for normal tissue are still far below the expectations with the severe adverse effects limiting their practical applications.11 The development of radioprotectants with high efficiency and low toxicity is highly challenging. With the fast developments of nanomedicine, the introduction of biocompatible theranostic nanoplatforms into radioprotection surpasses the aforementioned challenges. Typically, IR-induced injury is the consequence of undesirable pathological effect from free-radical species. Besides the functionality as drug carriers to improve the efficacy of molecular radioprotectors, nanoplatforms such as carbon-based, cerium-based, transition-metal dichalcogenide (TMDC) and noble metal nanosystems possess intrinsic capability of free radical scavenging.12 For instance, the water-soluble fullerenols are effective radioprotectors via eliminating free radicals and enhancing the antioxidative enzyme activities of total superoxide dismutases (SODs) and glutathione peroxidase (GPX),13 and their radioprotective efficacy is comparable to that of amifostine (AM), the first radioprotectant in clinical use.14-16 The single-layer graphene-encapsulated Fe and CoNi 3


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nanoshields (Fe@C and CoNi@C) manifest the radioprotective effects through scavenging reactive oxygen species (ROS) including superoxide anion (O2•-), hydroxyl (•OH), and hydroperoxyl (HO2•) free radicals.17 Graphdiyne-BSA nanoparticles can reduce the IR-induced intracellular ROS level and DNA damage.18 Cerium oxide nanoparticles display the radioprotective ability by inactivating the •OH and hydrogen peroxide (H2O2), shielding IR through the physical protective pathway and regulating some antioxidative enzymes as well as the proinflammatory cytokines.19-21 The cysteine-protected MoS2 nanodots achieve radioprotection through eliminating the free radicals by highly catalytic abilities toward H2O2 and oxygen-reduction reactions.22 Despite substantial progresses in the field of nanoradioprotectors, challenges remain in enhancing the biosafety/biodistribution and optimizing the physicochemical parameters of nanomaterials for higher efficiency in radiation protection. MXenes, a family of multifunctional two-dimensional (2D) ultrathin nanosheets (NSs) consisting of early transition metal and a large group of carbides, nitrides or carbonitrides, have recently attracted much attention in biomedical applications due to their distinctive physiochemical property and biological effect,23,24 and the applications of Ti3C2 in biosensors,25 antibacterial activity26 and photothermal therapy (PTT)27-30 have been explored. Especially, we successfully developed an ultrathin 2D niobium carbide (Nb2C) MXene as a photo-therapeutic agent in near-infrared (NIR)-II biowindow.31 Importantly, 2D Nb2C MXene possesses the intrinsic feature of enzyme/hydrogen peroxide (H2O2)-responsive biodegradability, which lowers the risk of adverse effects after therapeutic administration.31 Moreover, 2D MXene has the property in reacting with the generated free radicals. Therefore, it is reasonably speculated that ultrathin Nb2C MXene NSs can act as a potent radioprotectant with the concomitant antioxidant activity and biosafety. 4


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In this work, we employed ultrathin 2D Nb2C MXene as a radioprotectant to efficiently improve the survival of mice and reduce the normal-tissue damage especially in hematopoietic system in mice exposed to total body γ-irradiation (γ-TBI). The fabrication and intercalation of 2D ultrathin Nb2C NSs were achieved by a liquid exfoliation methodology. These Nb2C NSs were further surface-modified to produce Nb2C-polyvinyl pyrrolidone (noted as Nb2C-PVP) to improve the biocompatibility and physiological stability and reduce in vivo toxicity.31 The fabricated Nb2C-PVP NSs exhibit an excellent antioxidative performance with effective catalytic elimination of ROS including H2O2, O2•- and •OH, and show no noticeable cytotoxicity in vitro. The potential reaction mechanism of Nb2C-PVP in eliminating free radicals was revealed by theoretical calculation based on density functional theory (DFT). Furthermore, the in vitro and in vivo radiation-protection effects and the molecular mechanism were explored. Our studies demonstrate that Nb2C-PVP MXene NSs could exert the effects of radiation protection via the strong catalytic abilities in ROS scavenging. Especially, the 2D Nb2C-PVP displays highly in vivo radioprotective effects in hematopoietic tissue of mice exposed to γ-TBI. In particular, Nb2C-PVP is effectively cleared via liver and kidney and barely accumulated in the tissues 14 days after intravenous administration of Nb2C-PVP into the mice. Consistent with the results from our previous work,31 Nb2C-PVP at the effective radioprotection dose shows no obvious toxicities during 30-day post injection period.

RESULT AND DISCUSSION Fabrication and Characterization of Nb2C NSs (MXenes). Ultrathin Nb2C NSs were fabricated by a modified chemical exfoliation approach according to our previous report.31 HF 5


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aqueous solution was used to remove the middle Al layer of the MAX phase Nb2AlC. To achieve ultrathin Nb2C NSs, after HF etching, the Nb2C powder was delaminated in tetrapropylammonium hydroxide (TPAOH) aqueous solution. After sequential HF treatment and TPAOH intercalation, the bulk Nb2AlC ceramic was converted to 2D ultrathin MXenes (Figure 1a-b). From scanning electron microscopy (SEM) image, layered structure of the Nb2AlC MAX-phase ceramic was confirmed [Figure S1, Supporting Information (SI)]. From SEM image, the morphology of HF-etched Nb2C MXene was proved, which is in the form of multi-layer structure (Figure S2, 1c). After further TPAOH intercalation, well-dispersed Nb2C NSs could be obtained (Figure 1d). Bright-field and dark-field transmission electron microscopy (TEM) images reveal the single-layer structure of Nb2C NSs, which exhibit the typical planar structure with an average size of about 150 nm (Figure 1e-f). From selected area electron diffraction pattern (SAED), the hexagonal microstructure with high crystallinity of these 2D Nb2C MXenes was certified (Inset of Figure 1e). Energy dispersive spectroscopy (EDS) was used to analyze the detailed composition of Nb2C MXene.

From

Figure

1g,

a

high

angular

annular

dark

field

(HAADF)-spherical

aberration-corrected scanning transmission electron microscopy (STEM) image of Nb2C NSs was shown. The results exhibit that the carbon layer and niobium layers are alternately and evenly arranged. Nb2C NSs element distribution was examined by EDS mapping, which indicates that Nb2C NSs present a uniform distribution of Nb and C element from both side view (Figure 1h, S3) and surface view (Figure S4). From the X-ray diffraction (XRD) pattern, the bulk ceramic that has been completely converted into MXenes was implied. After etching, the (002) peak of Nb2C NSs (red curve) broadened and converted to a lower 2θ angle of 7.78°. The low-angle (002) peak as the most representative feature of MXenes changed from 39° to 7.78°, which implies that the bulk 6


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ceramic has been completely changed to MXene (Figure S5).32,33 From the result of atomic force microscopy (AFM), the morphology and size of Nb2C NSs was exhibited (Figure 1i, 1l). The thickness of Nb2C NSs is about 0.5 - 1 nm, which is identical to the thickness of the reported single and double layer Nb2C NSs (Figure 1j).31 The average size is statistically to be around 150 nm, which matches well with the TEM observation (Figure 1k). The N2 absorption-desorption isotherm-determined that Nb2C NSs process specific surface area of 2.977 m2/g (Figure S6). Therefore, these Nb2C NSs are characterized by lateral nano-size and ultra-thin nanostructures, making it feasible for further biomedical applications.

Mechanism of Radical-Scavenging Reactions of Nb2C NSs. The radical-clearance capability and potential mechanism of Nb2C NSs were deciphered using the density functional theory (DFT) calculation. The primitive crystal structure of exfoliated single-layered Nb2C was obtained by directly removing the Al layer from hexagonal Nb2AlC ceramic structure (materials id. mp-996162, P63/mmc). The two interlaced Nb layers form a sandwiched structure with the C layer along the z-axis. Sharing the C atom corners, six Nb atoms form two explicit head-to-head tetrahedrons, exposing the Nb layer on each side with carbon layer at the caves. The geometrically optimized structure of single-layered Nb2C (*) possesses a total-energy of -258.30 eV with uniform Nb-C bonding of 2.161 Å (Figure 2a-c). To mimic the IR-induced extreme oxidative stresses, we employed the •OH to assay the surface-structure revolution systematically. It has been found that the [Nb3C] site is the most energy-favorable adsorption site for the •OH after comparing [CNb3] site and top Nb atom site as 7


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indicated in Figure S7. The corresponding adsorption energy is then calculated to be -5.06 eV, which is assigned as moderate chemical binding. Such a process slightly distorts the original tetrahedron with Nb-O bonding of 2.255 - 2.260 Å and O-H bonding of 0.974 Å. From the investigated lattice, eight [Nb3C] sites are present on two sides along the z-axis. Upon fierce radical attack, the available [Nb3C] sites are gradually occupied. With generally similar adsorption energies, the energy of the system reduces sustainably until full [Nb3C] occupation (Figure 2d-e). The abundant exposure of surface Nb-OH species subsequently provides effective scavenging sites against the upcoming •OH by strong hydrogen-bonding interactions. The formation and subsequent desorption of H2O molecule lead to a net energy reduction of 3.6 eV, forming 7OH-O* species (Figure 2f). As the reaction proceeds, the surface hydroxyl groups can be effectively dehydrated upon continuous radical attack, forming additional O layer above the Nb layer (NbOx species) (Figure 2g-i). The ROS clearance property of Nb2C-PVP was originated from the frequent surface attack by •OH, forming oxygenic nanosheets, as supposed by the DFT calculations. In the X-ray photoelectron spectroscopy (XPS) spectrum of Nb, two peaks located at 205.4 and 203.7 eV were deconvoluted to the binding energies of Nb-C 3d3/2 and Nb-C 3d5/2 and the other two peaks located at 210.0 and 207.5 eV are corresponded to Nb2O5 3d3/2 and Nb2O5 3d5/2 (Figure 2j-l). Figure 2m are statistical graphs of different valence states of Nb before X-ray radiation, after 5 Gy and 10 Gy X-ray radiation in the Nb2C NSs. As the X-ray dose increases, the Nb-C species decreases, while the oxidized Nb2O5 species increases. Under 10 Gy X-ray irradiation, 28.34 % of Nb2C was oxidized into Nb2O5. Therefore, it is deduced that the reducibility of Nb2C NSs can reduce the ROS caused by IR. Since O2•- is one of the most destructive ROS generated during IR, we further examined the valence-state change of Nb2C NSs before and after the radical attack. O2•8


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is produced by the reaction of xanthine and xanthine oxidase.34 In the presence of O2•-, two peaks of NbC 3d3/2 (205.4 eV) and NbC 3d5/2 (203.7eV) disappeared, which were converted into the peaks of Nb2O5 3d3/2 (210.0 eV) and Nb2O5 3d5/2 (207.5 eV) entirely (Figure S8). The results demonstrate that Nb2C NSs were oxidized into Nb2O5 after reacting with O2•-. The XRD result was used to further verify this assumption. After the O2•- treatment, the Nb2C peak (black curve) changed. The newly appeared peak toward a 2θ angle of 25° implies the existence of Nb2O5 (Figure S9).35 The XPS and XRD results depict consistent results to the DFT calculations, revealing that the surface oxidation process is counted for the general ROS clearance capability. On the basis of experimental and calculation results, the mechanism of Nb2C NSs for scavenging free radicals generated by IR could be attributed to the intrinsic reducing property of MXene nanosheets, which are further oxidized into Nb-based oxides by scavenging ROS.

Characterization of the Radical-Scavenging Performance of 2D Nb2C-PVP. 2, 2’-azino-bis 3-ethylbenzthiazoline-6-sulfonic acid (ABTS) is commonly used as a color developer to test the capacity of the antioxidant. ABTS is oxidized to green ABTS•+ under the addition of an appropriate oxidant, and ABTS•+ is reduced in the presence of antioxidants. The total antioxidant capacity of Nb2C-PVP was determined and calculated by measuring the absorbance of ABTS•+ at 414 nm in UV-vis spectrum. Trolox is an analog of vitamin E, which has similar antioxidant properties to vitamin E, and serves as a reference.36 It was found that Nb2C-PVP significantly increased the values of Trolox-Equivalent Antioxidant Capacity (TEAC) in a concentration-dependent manner, indicating that Nb2C-PVP is featured with strong antioxidant capacity (Figure 3a).

9


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The cyclic voltammetric (CV) test was used to detect the catalytic abilities of Nb2C-PVP via H2O2 elimination and oxygen reduction reactions (ORR). In the presence of 0.01 M PBS solution (O2-saturated), the as-prepared Nb2C-PVP working electrode achieves larger negative current density at the potential of -0.7 V compared to the glassy carbon (GC) electrode, indicating that Nb2C-PVP possesses the catalytic activity of ORR (Figure 3b). Figure 3c shows the CV current of Nb2C-PVP measured in H2O2 (N2-saturated). Similarly, larger negative current density at the potential of -0.7 V was observed in the presence of Nb2C-PVP compared to unmodified GC electrode, manifesting that Nb2C-PVP has the ability towards H2O2 elimination. Therefore, the results of the CV curve imply the extraordinarily catalytic abilities of Nb2C-PVP towards H2O2 and O2 reduction. It is well known that O2•- and •OH are highly destructive ROS, which can be generated by IR and induce damages of DNA, protein, and lipid in the cells.37,38 The effect of Nb2C-PVP on depleting O2•- was tested by the nitroblue tetrazolium (NBT) method. While O2•- reduces the NBT to blue formazan with strong absorption at 560 nm, SODs can inhibit the formation of formazan by eliminating O2•-. As shown in Figure 3d, when the concentration of Nb2C-PVP was 80 µg/mL, 100 µg/mL and 120 µg/mL, the O2•- elimination of Nb2C-PVP reached 33.01%, 51.16%, and 71.97%, respectively,

which

indicates

that

Nb2C-PVP

could

effectively

scavenge

O2•-

in

a

concentration-dependent way. It can be deduced that Nb2C-PVP has a SODs-like activity. The •OH-scavenging activity of Nb2C-PVP was further evaluated by the electron spins resonance (ESR) measurement. The •OH was generated by the Fenton reaction with the Fe2+/H2O2 system. The spin trap 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) was used to form BMPO/•OH adducts. As shown in Figure 3e, the BMPO/ •OH signal (four characteristic peaks with intensities 10


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of 1:2:2:1) decreased with the elevation of the Nb2C-PVP concentration, indicating the Nb2C-PVP capability to eliminate •OH.

Radiation Protection by Nb2C-PVP In Vitro. The high biocompatibility and low in vitro and in vivo toxicity of Nb2C-PVP have been demonstrated in our previous work exploiting Nb2C-PVP as a photothermal therapeutic agent of tumors.31 Here, for further exploring the bioapplication of Nb2C-PVP as a radioprotectant, we first examined the in vitro toxicity of Nb2C-PVP in normal cells using a standard CCK-8 assay. It was found that Nb2C-PVP treatment at concentrations up to 200 µg/mL for 24 h and 48 h was not cytotoxic to mouse embryonic fibroblast 3T3/A31 cells (Figure 4a), which is consistent with our previous study indicating that Nb2C-PVP at the same concentration was not cytotoxic to 4T1 breast cancer cells and U87 glioma cells.31 We then assessed the effects of non-cytotoxic Nb2C-PVP on the cell survival of 3T3/A31 cells exposed to IR at different doses. As expected, pre-treatment of Nb2C-PVP at concentrations of 50 and 100 µg/mL for 24 h was able to significantly rescue 3T3/A31 cell death induced by X-ray radiation (Figure 4b). The result was further confirmed by the Calcein-AM/PI cell staining assay where the living cells and the nucleus of the dead cells were stained with green and red colors, respectively (Figure 4c). Peripheral blood lymphocytes (PBL) are known to be extremely sensitive to IR.2 We isolated lymphocytes from rat peripheral blood and examined PBL apoptosis after X-ray radiation with or without Nb2C-PVP pre-treatment using the TdT mediated dUTP nick end labeling (TUNEL) staining. While Nb2C-PVP treatment alone had no effects on PBL apoptosis, Nb2C-PVP pre-treatment significantly decreased the TUNEL positive cells induced by X-ray radiation (Figure 4d-e). Furthermore, Nb2C-PVP pre-treatment significantly decreased the level of IR-induced ROS 11


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in 3T3/A31 cells (Figure 4f), suggesting that the radioprotective ability of Nb2C-PVP is associated with its effective free radical-scavenging activity. A prerequisite of radioprotectants for protecting cancer patients from severe side effects during radiotherapy is the radioprotective effects occurring in normal tissues but not in tumors. We further assessed the effects of Nb2C-PVP on irradiated cancer cells including HepG2 liver cancer cells, A549 lung cancer cells and 4T1 breast cancer cells. Figure S10 shows that pre-treatment of Nb2C-PVP at 50 and 100 µg/mL for 4 h or 24 h had no radioprotective effects on all the tested cancer cells. It has been reported that cerium oxide nanoparticles, Fe@C and CoNi@C nanoshields show a significantly radioprotective efficacy in normal cells but not in cancer cells.17,39 The cellular selectivity of Nb2C-PVP in radiation protection may be attributed to the lower level of H2O2 in normal cells,40 resulting in the lower degradation rate of Nb2C-PVP in normal cells than that of tumor cells,31 as well as the more relaxed chromatin structure as ROS targets in cancer cells than in normal cells.39

Nb2C-PVP Enhances Mouse Survival and Promotes Hematopoietic Recovery after IR. To determine whether Nb2C-PVP is stable in vivo, we initially examined Nb2C-PVP stability in simulated body fluid (SBF). Therefore, we suspended Nb2C-PVP in SBF under the physiological condition. The morphology and microstructure evolution of Nb2C-PVP was systematically observed under TEM at varied time intervals. The O and Nb element contents of Nb2C-PVP were in-situ determined by EDS. Figure S11 shows that there were no significant changes of morphology/microstructure and oxygen content of Nb2C-PVP within 24 h, indicating that 12


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Nb2C-PVP possesses relatively high stability under the physiological condition. Next, we studied the effects of Nb2C-PVP pre-treatment at different doses on survival of irradiated mice. BALB/C mice were injected intravenously with Nb2C-PVP in PBS at 5, 10 or 20 mg/kg 24 h before γ-TBI at sub-lethal (5 Gy) or lethal (6.5 Gy) doses.41 At the sub-lethal radiation dose (5 Gy TBI), the 30-day survival rate was 100% in the mice with 20 mg/kg Nb2C-PVP pre-treatment as compared to 27% in the mice receiving the vehicle only with the median survival time of 21 days and a 95% confidence interval (CI) of 18 - 25 days (Figure 5a). Moreover, at the lethal dose (6.5 Gy) of TBI, the 30-day survival rate was 30% in the mice with 5 mg/kg Nb2C-PVP pre-treatment with the median survival time of 21 days and a 95% CI of 18 - 25 days, and 50% in the mice with 10 mg/kg Nb2C-PVP pre-treatment with the median survival time of 24 days and a 95% CI of 21 - 28 days, and 81% in the mice with 20 mg/kg Nb2C-PVP pre-treatment with the shortest survival time of 21 days as compared to IR alone mice with 100% mortality and the mean survival time of 15 days (95% CI: 14 - 16 days) (Figure 5a). For comparison, pre-treatment of amifostine (AM) at a dose of 400 mg/kg, the most high efficacy dose,42 30 min before TBI provided 90% survival with the shortest survival time of 20 days in mice after TBI of 6.5 Gy, which was similar in mice with 20 mg/kg Nb2C-PVP pre-treatment plus 6.5 Gy TBI group (Figure 5a). Recently, it has been reported that Fe@C, CoNi@C nanoshields and cysteine-protected MoS2 nanodots exhibited higher efficiency in radiation protection, which enhanced the surviving fractions of mice exposed to lethal TBI up to 90%, 80% and 79%, respectively.17,22 Taken together, Nb2C-PVP possesses significant radioprotective activity in vivo, which is comparable to that of the standard radioprotectant AM and several potent nanoradioprotectors, and a dose of 20 mg/kg Nb2C-PVP is recommended. 13


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Hematopoietic system is extremely sensitive to IR, and radiation death with sub-lethal dose IR is associated with hematopoietic failure. Therefore, the recovery of IR-induced hematopoietic system injury is critical for survival and quality of life after exposure.43 IR-induced injury in hematopoietic system is characterized by BM suppression, exhibiting loss of the BM cellularity mainly consisting of hematopoietic stem cells and hematopoietic progenitor cells, where mitotic death from IR-induced cytogenetic damage contributes primarily to IR-induced lethality in the different hematopoietic compartments.37,44 Accordingly, we evaluated the radioprotective effects of Nb2C-PVP on IR-induced hematopoietic system injury by measuring the following parameters: histopathology of bone marrow (BM), counts of bone marrow mononuclear cells (BM-MNCs) and micronuclei of polychromatic erythrocytes (MN-PCE) and hematological parameters at 1, 7, and 30 days after 5 Gy TBI, and count of endogenous spleen colony-forming units (CFU-S) at 7 days after 6.5 Gy TBI. As shown in Figure 5b, after 5 Gy irradiation, BM-MNCs contents in the medullary cavity were significantly reduced and fully replaced with red blood cells due to destruction of BM vasculature, especially 7 days post-TBI (IR alone group), when the pathological changes of the BM entered a stage of severe emptiness. Pre-treatment with Nb2C-PVP alleviated the IR-induced BM depletion and hemorrhage to a large extent by increasing the amount of BM-MNCs and decreasing the number of red blood cells at 1, 7 and 30 days post-TBI, and especially, BM cell composition at 30 days TBI was almost similar to that in unirradiated normal mice. These data indicate that Nb2C-PVP accelerates hematopoietic recovery by enhancing hematopoiesis, thereby attenuates IR-induced BM damage. We further used BM-MNCs count to verify the pathological change of BM cellularity post-TBI (Figure 5c). At 1 and 7 days post-TBI, BM-MNCs count was significantly decreased to nearly 7% 14


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and 3%, respectively, in the IR alone mice compared to that of unirradiated control mice. In contrast, BM-MNCs count in the irradiated mice with pre-treatment of Nb2C-PVP was nearly 3 and 7 folds higher than those of IR alone mice at 1 and 7 days post-TBI. At day 30, the BM-MNCs count in the IR alone mice was still significantly lower than the level in unirradiated control mice, suggesting that the recovery was incomplete. On the contrast, BM-MNCs count in the irradiated mice pre-treated with Nb2C-PVP was much higher than that of IR alone group, and was close to the level in the unirradiated control mice, suggesting a complete recovery at 30 days post-TBI, which was consistent to the recovery of the histological injury of BM. IR-induced DNA lesions are not only manifested as chromosomal breaks/aberrations as a result of DNA double-strand breaks and misrepair but also displayed as micronuclei from lagging whole chromosomes and acentric chromosome fragments at anaphase. Micronuclei can be observed in the cytoplasm besides the cell nucleus as small nucleus-like particles. To quantify the level of DNA damage, MN-PCE assay was used. This assay has been shown to be an effective tool to measure IR-induced cytogenetic (chromosomal) damage in BM and to monitor mitotic death in IR-induced depletion of BM.45 As shown in Figure 5d-e, the MN-PCE frequency was significantly increased in irradiated mice, which was nearly 1.8 and 2.4 folds higher than that in unirradiated control mice at 1 and 7 days, respectively, after 5 Gy TBI, indicating that the BM DNA in the irradiated mice was damaged. In contrast, administration of Nb2C-PVP markedly decreased the MN-PCE frequency, which was nearly 43% and 40% of the values seen in irradiated mice at 1 and 7 days, respectively, after 5 Gy TBI. Importantly, the MN-PCE frequency in Nb2C-PVP pre-treatment plus IR group was reduced to a normal level in unirradiated control mice at 7 days after 5 Gy TBI. These data indicate that Nb2C-PVP effectively reduces the IR-induced cytogenetic damage to mouse BM cells. 15


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To determine the radioprotective effects of Nb2C-PVP on IR-induced hematopoietic stem-cell injury, we performed the endogenous colony-forming units (CFU) assay. The number of CFU-S is an indicator of hematopoietic function and important marker of hematopoietic recovery.46,47 After lethal dose (6.5 Gy) irradiation, a small number of spleen nodules (2.50 ± 1.05/spleen) were formed in the spleen of IR alone group, suggesting that a small amount of hematopoietic stem cells still survived in the body. Irradiated mice pre-treated with Nb2C-PVP formed a large number of spleen nodules (11.17 ± 3.43/spleen) in the spleen, which was increased by nearly 5 folds compared to TBI alone group (Figure 5f), suggesting that Nb2C-PVP has the ability to reduce IR-induced impairments of hematopoietic stem cells’ self-renewal capacity, reconstitute myeloid cells and promote the recovery of the hematopoietic system. IR induces hematopoiesis damage, resulting in the depletion of all lineages of peripheral blood cells.43 We then used peripheral blood cell count to assess the radioprotective effect of Nb2C-PVP on IR-induced BM suppression. As shown in Figure 5g-i, the sublethal dose TBI caused decreases of all three types of hemocytes. WBC count sharply decreased nearly 70% and 97% in the irradiated mice compared to those of unirradiated control mice at 1 and 7 days post-TBI, respectively, and was still markedly lower than those in unirradiated control mice though it elevated at 30 days post-TBI. RBC and PLT count decreased slowly and decreased nearly 30% and 59% in the irradiated mice compared to those in unirradiated control mice at 7 days post-TBI, which were markedly lower than those of unirradiated group. In contrast, pre-treatment with Nb2C-PVP significantly enhanced the WBC count, which were nearly 2 and 3 folds higher than those in IR alone mice at 1 and 7 days post-TBI. Moreover, Nb2C-PVP stimulated the recovery of WBC, which returned to the normal level at 30 days post-TBI, as compared with the unirradiated control group. Reduction of the 16


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decrease of RBC and PLT was found in mice pre-treated with Nb2C-PVP at 7 days post-TBI. These data indicate that pre-irradiation administration of Nb2C-PVP not only protects peripheral hematocytes from IR-induced damage but also stimulates hematopoietic recovery of BM.

Nb2C-PVP Protects against IR-Induced Multiple Organ Degeneration. In addition, the efficacy of Nb2C-PVP in the protection of various tissues including testis, small intestine, lung, and liver in mice irradiated with sub-lethal dose (5 Gy) of γ-rays was examined. The testis is highly sensitive to IR, and germ cells can be damaged after IR exposure as low as 0.1 Gy.48,49 The pathohistological examination of testes of irradiated mice showed a severe degeneration (vacuolation) and decreased a number of spermatogenic cells in the seminiferous tubule at 7 days after irradiation. The phenomenon of spermatogenic cells in the testes of the Nb2C-PVP pre-treated mice was restored, and the morphology was almost normal. The small intestine of irradiated mice exhibited the mild to moderate intensity of pathohistological changes including partial villous atrophy, loose arrangement and abnormal crypts with an increase in size and decrease in number. The abnormal crypts were likely resulted from retarded regeneration after IR-induced crypt epithelial cells damages. No obvious intestinal alteration was found in irradiated mice protected with Nb2C-PVP. Irradiation of lungs caused moderate pulmonary lesions. The scattered alveolar edema and interstitial pneumonia with alveolar wall thickening and leukocytic infiltration were observed. A small number of hemorrhagic foci were found in the pulmonary interstitium. However, the changes in lung tissues in the Nb2C-PVP pre-treated mice were not obvious. The examination of the liver of irradiated mice showed extensive hydropic degeneration (cellular swelling) with vacuolation and hypertrophied hepatocytes containing condensed nucleus in the hepatocytes. 17


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Nb2C-PVP pre-treatment resulted in an improvement in hepatocyte morphology. These pathological results demonstrate that damage of testis, small intestine, lung, and liver of irradiated mice could be reduced, even no obvious abnormalities were observed by pre-treatment with Nb2C-PVP (Figure 6).

Nb2C-PVP Restores SODs Activities and Suppresses MDA Level after IR In Vivo. IR-induced ROS causes oxidative damage in cells and tissues via attacking biological macromolecules such as nucleic acids, lipids and proteins,37,38 the superoxide dismutases (SODs) constitute the first line of defense against ROS.38 Malondialdehyde (MDA) is an end-product of oxidative degradation of lipids, and its level reflects the extent of cell membrane damage. To further elucidate the protection mechanism of the Nb2C-PVP in vivo, we examined the effects of Nb2C-PVP pre-treatment on IR-induced oxidative stress by measuring the ROS level or total SODs activities and MDA content in hematopoietic tissue, testes, small intestine, lung tissues and liver in mice exposed to sub-lethal TBI. As shown in Figure 7a-b, exposure of mice to a sub-lethal dose of TBI induced a significant increase of ROS production and a distinct decrease of total SODs activities in BM-MNCs. It was found that ROS levels in irradiated mice at 1, 7 and 30 days post-TBI was 1.74, 1.63 and 1.27 times higher than that in healthy mice, while total SODs activities in irradiated mice was 95%, 97%, and 46% lower than that in healthy mice, indicating that excessive ROS represses the activities of SODs, resulting in impaired ability of self-protection of mice. Compared with IR alone group, pre-treatment of irradiated mice with Nb2C-PVP significantly reduced or even prevented the IR-induced production of ROS and significantly enhanced total SODs activities in BM-MNCs, showing that the level of ROS at 1, 7 and 30 days post-TBI and total 18


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SODs activities at 30 days post-TBI were close to those in BM-MNCs of unirradiated mice. As shown in Figure 7c-j, exposure to TBI also induced 38% ~ 71% decreases in total SODs activities and 1.6 ~ 2.8-fold increases in MDA levels in testes, small intestine, lung tissues and liver at 1 and 7 days post-TBI. In contrast, pre-treatment with Nb2C-PVP significantly enhanced the total SODs activities and decreased the MDA contents in these tissues at 1 and 7 days post-TBI, and some of them were almost comparable to the levels of healthy mice. These results indicate that the Nb2C-PVP reduces the IR-induced suppression of SODs activities and MDA levels via scavenging IR-induced ROS. Pharmacokinetics, Biodistribution and Metabolism of Nb2C-PVP. Generally, the slower metabolism of nanomaterials in vivo than that of small molecular compounds is one of their important characteristics. Accordingly, nanomaterials exhibit longer-term in vivo effects.22,50,51 On the other hand, it may also increase the unwanted toxicity.52-55 Therefore, the pharmacokinetics, biodistribution and metabolism profiles of Nb2C-PVP are not only important to assess the biosafety but also can help to determine an optimal time of Nb2C-PVP administration to maximize its in vivo radioprotective effects. We first investigated the pharmacokinetics of Nb2C-PVP at the dose of 20 mg/kg in BALB/C mice. The results show that Nb2C-PVP with intravenous administration has a plasma half-life of 3.8 h in BALB/C mice (Figure 8a). The cysteine-protected MoS2 nanodots, an effective radioprotectant, has also been found to manifest a long blood circulation with a plasma half-life of 2.1 h.22 It is clear that Nb2C-PVP overcomes the defect of AM with a very short plasma half-life less than 10 min,56 which limits its radioprotection ability in vivo.57-59 The tissue distribution of Nb2C-PVP shows that Nb existed in almost all tissues including liver, lung, spleen, heart, kidney, testis and small intestine at 4 - 48 h after intravenous injection of 19


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Nb2C-PVP at 5 and 20 mg/kg into BALB/C mice (Figure 8b, Figure S12a), which is consistent with our previous results in immunodeficient nude mice.31 Critically, Nb content was also detected in BM and BM-MNCs isolated from two femurs (Figure 8c, Figure S12b). The characteristic of non-selective distribution in tissues/organs is advantageous for Nb2C-PVP as a radioprotectant, as Nb2C-PVP could display protective effects for all tissues/organs exposed to IR. Several studies on nanoradioprotectors reported a similar observation.22,60 However, Nb2C-PVP with the intravenous administration showed an uniform distribution in different tissues/organs. Figure 8b shows that there was a relatively higher Nb content in liver than other tissues/organs after 4 - 48 h injection, which is consistent with our previous results in immunodeficient nude mice.31 Notably, the rates of liver uptake and clearance of Nb2C-PVP were higher than those of other tissues/organs after 4 - 48 h injection, suggesting that Nb2C-PVP is primarily metabolized in liver. It is considered that the large nanomaterials (> ~200 nm) can be quickly uptaken and degraded by Kupffer cells in the liver into smaller ones, and nanoparticles smaller than the diameter (~ 100 – 200 nm) of fenestration between the endothelium of hepatic sinusoid can be uptaken by hepatocytes and subsequently cleared by the biliary excretion pathway.50,61 Importantly, nearly all tissues including BM, liver, lung, spleen, heart, kidney, testis and small intestine reached the maximum accumulation of Nb at 24 h after intravenous injection of Nb2C-PVP into BALB/C mice (Figure 8b, c and Figure S12). It was reported that on the first day after PVP-protected Bi2Se3 nanoplates (54 nm in width) were intraperitoneally injected into C57 male mice, both Bi and Se were highly uptaken in all tissues.55 Therefore, it is appropriate to choose intravenous administration of Nb2C-PVP into the mice 24 h before IR for radioprotection. This also means that Nb2C-PVP in the tissues/organs requires to reach a certain amount before it plays a role in radioprotection. It is well-known that the 20


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radiosensitivity is cell type- and tissue type-dependent.2 Hematopoietic system is highly sensitive to IR, and IR-induced hematopoietic failure with hematopoietic stem/progenitor cell injury is the primary cause of death after exposure to sub-lethal dose of TBI.37 Therefore, we further examined the Nb content in BM-MNCs isolated from BM during 4 - 48 h after injection of Nb2C-PVP at 5 mg/kg, the lowest effective dose of Nb2C-PVP for protection mice exposed to 6.5 Gy lethal dose irradiation, to explore the minimum amount of Nb2C-PVP required to provide effective protection for BM at the cellular level. Figure S12b shows that Nb content in BM-MNCs at 24 h after 5 mg/kg Nb2C-PVP administration is 0.045 pg/BM-MNC, which could be the minimum effective amount for protecting against IR-induced BM-MNCs injury. Since tissues/organs consist of a variety of cells with different radiosensitivity,2 it is difficult to determine the minimum amount of Nb2C-PVP required for effective protection for different tissues/organs at the tissue level. Anyway, the minimum amount of Nb2C-PVP required for protecting the cells with moderate and low radiosensitivity in the tissues/organs should be lower than it. Therefore, the dosage of Nb2C-PVP should be adjusted to protect each individual tissue from local irradiation. In our previous study, we found that Nb content in normal tissues decreased with time while tumor tissue reached the maximum accumulation of Nb after 24 h of Nb2C-PVP intravenous injection in tumor-bearing nude mice.31 The time-related biodistribution difference of Nb2C-PVP in normal tissues between healthy BALB/C mice and tumor-bearing nude mice may be related to the difference between normal mice and immunodeficient nude mice or targeted enrichment of Nb2C-PVP in tumor tissue.31 It is noteworthy that Nb element but not Nb2C-PVP in tumor tissue was detected in our previous work.31 It is reasonable to speculate that Nb2C-PVP in the tumor tissues could be actually transformed to the Nb-based oxides within the high H2O2 level 21


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environment of tumor cells, resulting in no radioprotection on tumor cells, though Nb content in tumor tissue reached the maximum at 24 h post injection. Indeed, there were no radioprotective effects of Nb2C-PVP pre-treatment for 4 and 24 h on in vitro irradiated tumor cells (Figure S10). As we expected, Nb2C-PVP could be metabolized efficiently in all tissues. The Nb content in tissues decreased by 27.9% - 71.7% with the highest decrease rate in the liver at 48 h post injection, 83.6% - 99.0% on day 7 post injection, and 97.1% - 99.9% on day 14 post injection compared to 24 h post injection upon treatment with Nb2C-PVP at 20 mg/kg (Figure 8b, c). More specifically, at 48 h post injection, residual Nb in the liver and spleen was decreased to 10.2% and 4.6% of injected dose (ID) /g (tissues), which corresponded to 40.8 and 18.4 μg/g (tissues) while residual Nb in lung, heart, kidney, testis and small intestine was decreased to 0.1% - 4.8% of ID/g (tissues), which corresponded to 0.4 – 19.2 μg/g (tissues); residual Nb in the BM was decreased to 0.35% of ID/BM of femurs, which corresponded to 0.05 pg/BM-MNC. On day 7 post injection, residual Nb in the liver and spleen was decreased to 1.2% and 1.4% of ID/g (tissues), which corresponded to 4.8 and 5.6 μg/g (tissues) while residual Nb in lung, heart, kidney, testis and small intestine was decreased to 0.03% - 0.62% of ID/g (tissues), which corresponded to 0.12 - 2.48 μg/g (tissues); residual Nb in the BM was decreased to 0.02% of ID/BM of femurs, which corresponded to 0.002 pg/BM-MNC. On day 14 post injection, residual Nb in the liver and spleen was decreased to 0.09% and 0.24% of ID/g (tissues), which corresponded to 0.36 and 0.96 μg/g (tissues) while residual Nb in lung, heart, kidney, testis and small intestine was decreased to 0.005% - 0.067% of ID/g (tissues), which corresponded to 0.020 - 0.268 μg/g (tissues); residual Nb in the BM was decreased to 0.002% of ID/BM of femurs, which corresponded to 0.0005 pg/BM-MNC, indicating that Nb2C-PVP was almost completely excreted from the body. Moreover, nearly 20% of Nb could be excreted through 22


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feces and urine (Figure 8d) during 48 h post injection in BALB/C mice, which is consistent with the results of tumor-bearing nude mice.31 Nearly 80% of Nb could be excreted through feces (57%) and urine (23%) during 7 days post injection (Figure 8d). Zhang et al. reported that nearly 80% of ultrasmall cysteine-protected MoS2 nanodots (

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