(MXene) Promotes Hematopoietic Recovery after ... - ACS Publications

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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*,† Downloaded via UNIV OF SOUTHERN INDIANA on July 19, 2019 at 06:28:11 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Department of Radiation Biology, Institute of Radiation Medicine, Fudan University, Shanghai, 200032, People’s Republic of China State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, People’s Republic of China § University of Chinese Academy of Science, Beijing, 100049, People’s Republic of China ‡

S Supporting Information *

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 damage to normal tissues. In this work, we have developed ultrathin twodimensional (2D) niobium carbide (Nb2C) MXene as a radioprotectant and explored its application in scavenging free radicals against IR. The 2D Nb2C MXene features intriguing antioxidant properties in effectively eliminating hydrogen peroxide (H2O2), hydroxyl radicals (•OH), and superoxide radicals (O2•−). Pretreatment with biocompatible polyvinylpyrrolidone (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 and 6.5 Gy irradiated mice to 100% and 81.25%, respectively, and significantly increases bone marrow mononuclear cells after IR. Critically, Nb2C-PVP reverses the damage of the hematopoietic system in irradiated mice. Single administration of Nb2C-PVP significantly increases superoxide dismutase (SOD) activities, decreases malondialdehyde 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 dismutase, hematopoiesis

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use, and their radioprotective efficiencies for normal tissue are still far below 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 an

ith 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 use. Over the years, researchers have been dedicated to developing effective radioprotectors via various strategies including free radical scavenging, antioxidation, immune stimulation, and Toll-like receptor agonist.6−10 However, very few radioprotectors have been developed for clinical © 2019 American Chemical Society

Received: December 10, 2018 Accepted: June 3, 2019 Published: June 3, 2019 6438

DOI: 10.1021/acsnano.8b09327 ACS Nano 2019, 13, 6438−6454

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Cite This: ACS Nano 2019, 13, 6438−6454

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Figure 1. Fabrication and characterization of 2D ultrathin Nb2C NSs (MXene). (a) Schematic diagram for the fabrication of ultrathin Nb2C NSs, including stepwise HF etching and TPAOH intercalation. (b) Scheme of the exfoliation process for Nb2C NSs based on a ball-and-stick model. (c) SEM image of multilayer Nb2C. (d) Digital photograph, (e) bright-field TEM image (inset shows the original SAED pattern), and (f) dark-field TEM image of single-layer Nb2C. (g) High-resolution HAADF-STEM and (h) EDS elemental mapping of few-layer Nb2C NSs from a side view (red and green colors represent carbon and niobium, respectively). (i) Typical AFM image of Nb2C NSs. (j, k) Thickness distribution and lateral size of Nb2C NSs. (l) 3D AFM view of the area in panel i.

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 6439


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displays high in vivo radioprotective effects in hematopoietic tissue of mice exposed to γ-TBI. In particular, Nb2C-PVP is effectively cleared via the 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 the 30-day postinjection period.

(TMDC), and noble metal nanosystems possess an intrinsic capability of free radical scavenging.12 For instance, the watersoluble fullerenols are effective radioprotectors via eliminating free radicals and enhancing the antioxidative enzyme activities of total superoxide dismutase (SOD) 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 grapheneencapsulated Fe and CoNi nanoshields (Fe@C and CoNi@C) manifest 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 a radioprotective ability by inactivating the •OH and hydrogen peroxide (H2O2), shielding IR through a 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 free radicals by highly catalytic abilities toward H2O2 and oxygen-reduction reactions.22 Despite substantial progress 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 an 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 activity,26 and photothermal therapy (PTT)27−30 have been explored. Especially, we successfully developed an ultrathin 2D niobium carbide (Nb2C) MXene as a phototherapeutic agent in the near-infrared (NIR)-II biowindow.31 Importantly, 2D Nb2C MXene possesses the intrinsic feature of enzyme/H2O2-responsive biodegradability, which lowers the risk of adverse effects after therapeutic administration.31 Moreover, 2D MXene has the property of reacting with the generated free radicals. Therefore, it is reasonably speculated that ultrathin Nb2C MXene NSs can act as a potent radioprotectant with concomitant antioxidant activity and biosafety. 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 a 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 Nb2Cpolyvinylpyrrolidone (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 radiationprotection 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

RESULTS AND DISCUSSION Fabrication and Characterization of Nb2 C NSs (MXenes). Ultrathin Nb2C NSs were fabricated by a modified chemical exfoliation approach according to our previous report.31 A hydrofluoric acid (HF) 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 a 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 a scanning electron microscopy (SEM) image, the layered structure of the Nb2AlC MAX-phase ceramic was confirmed [Figure S1, Supporting Information (SI)]. From a SEM image, the morphology of HF-etched Nb2C MXene was proved, which is in the form of a multilayer structure (Figures 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 a typical planar structure with an average size of about 150 nm (Figure 1e,f). From the 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. In Figure 1g, a high angular annular dark field (HAADF)-spherical aberrationcorrected scanning transmission electron microscopy (STEM) image of Nb2C NSs is shown. The results exhibit that the carbon layers 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 elements from both the side view (Figures 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 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 were exhibited (Figure 1i,l). 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 around 150 nm, which matches well with the TEM observation (Figure 1k). The N2 absorption−desorption isotherm determined that Nb2C NSs possess a specific surface area of 2.977 m2/g (Figure S6). Therefore, these Nb2C NSs are characterized by lateral nanosize and ultrathin nanostructures, making it feasible for further biomedical applications. Mechanism of Radical-Scavenging Reactions of Nb2C NSs. The radical-clearance capability and potential mechanism 6440


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Figure 2. Radical-scavenging reaction of Nb2C NSs. (a−c) Geometrically optimized single-layer Nb2C nanostructure viewed in (a) orthogonal, (b) front, and (c) left side. (d) Optimized structures of Nb2C NSs upon exposure to •OH. (e) Total energy and the adsorption energy of each species during the •OH attack of Nb2C NSs. Inset indicates the radical-scavenging site. (f) Energy diagram of the system during the further oxidation of Nb2C NSs by •OH upon the surface −OH dangling site. (g−i) Geometrically optimized full nanostructure of oxidized Nb2C viewed in (g) orthogonal, (h) front, and (i) left sides. XPS spectra of Nb2C NSs in the Nb 3d region (j) before X-ray radiation and after (k) 5 and (l) 10 Gy X-ray radiation on Nb2C NSs. (m) Statistical graphs of different valence states of Nb before X-ray radiation and after 5 and 10 Gy X-ray radiation on Nb2C NSs.

systematically. It has been found that the [Nb3C] site is the most energy-favorable adsorption site for •OH after comparing the [CNb3] site and top Nb atom site as 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 a 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 strong radical attack, the available [Nb3C] sites are gradually occupied. With generally similar adsorption energies, the energy of the system decreases sustainably until full [Nb3C] occupation (Figure 2d,e). The abundant exposure of surface Nb−OH species subsequently

of Nb2C NSs were deciphered using DFT calculations. The primitive crystal structure of exfoliated single-layered Nb2C was obtained by directly removing the Al layer from the 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 tetrahedra, exposing the Nb layer on each side to the carbon layer at the caves. The geometrically optimized structure of single-layered Nb2C (*) possesses a total energy of −258.30 eV with a uniform Nb−C bonding of 2.161 Å (Figure 2a−c). To mimic the IR-induced extreme oxidative stresses, we employed •OH to assay the surface-structure revolution 6441


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Figure 3. Radical scavenging by Nb2C-PVP. (a) Antioxidant capacity of Nb2C-PVP measured by the ABTS method. (b) CVs of the GC electrode modified with as-prepared Nb2C-PVP in O2-saturated 0.01 M PBS (pH 7.4) and a pure GC electrode in 0.01 M PBS (pH 7.4). (c) CVs of the GC electrode modified with as-prepared Nb2C-PVP in the presence of 5.0 × 10−3 M H2O2 in N2-saturated 0.01 M PBS (pH 7.4) and bare GC electrode in PBS (H2O2). (d) O2•−-scavenging activity of Nb2C-PVP (80 μg/mL, 100 μg/mL, 120 μg/mL). (e) ESR spectra of a BMPO/•OH mixture as obtained from samples containing 4 × 10−3 M H2O2, 4 × 10−4 M FeSO4, and 25 mM BMPO. All the values are the average from three independent experiments with the SD indicated by error bars. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control group, #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the low-concentration group.

provides effective scavenging sites against the upcoming •OH by strong hydrogen-bonding interactions. The formation and subsequent desorption of H2O molecules 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 an additional O layer above the Nb layer (NbOx species) (Figure 2g−i). The ROS clearance property of Nb2C-PVP originated from the frequent surface attack by •OH, forming oxygenic nanosheets, as supported 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 correspond to Nb2O5 3d3/2 and Nb2O5 3d5/2 (Figure 2j−l). In Figure 2m are statistical graphs of different valence states of Nb before X-ray radiation and after 5 and 10 Gy X-ray radiation in the Nb2C NSs. As the X-ray dose increases, the Nb−C species decrease, while the oxidized Nb2O5 species increase. 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•− is produced by the reaction of xanthine and xanthine oxidase.34 In the presence of O2•−, two peaks of Nb−C 3d3/2 (205.4 eV) and Nb−C 3d5/2 (203.7 eV) 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 appearing peak toward a 2θ angle of 25° implies the existence of Nb2O5 (Figure S9).35 The XPS and XRD results depict consistent results with the DFT calculations, revealing that the surface oxidation process accounts 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-ethylbenzthiazoline6-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 the UV−vis spectrum. Trolox is an analogue 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 Nb 2 C-PVP features a strong antioxidant capacity (Figure 3a). 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 phosphate buffered saline (PBS) solution (O2-saturated), the as-prepared Nb2C-PVP working electrode achieves a larger negative current density at the potential of −0.7 V compared to the glassy carbon (GC) electrode, indicating that Nb2CPVP possesses the catalytic activity of ORR (Figure 3b). Figure 6442


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Figure 4. In vitro evaluation of radiation protection by Nb2C-PVP. (a) Relative viability of 3T3/A31 cells after incubation with Nb2C-PVP at varied concentrations (0, 6, 12, 25, 50, 100, and 200 μg/mL) for 24 and 48 h. (b) Relative viability of 3T3/A31 cells after exposure to IR at different doses (0, 3, 6, 9, and 12 Gy) with pretreatment of Nb2C-PVP at different concentrations (0, 50, and 100 μg/mL). (c) Representative fluorescence images of X-ray radiation (6 Gy)-induced cell death after various treatments (control, IR only, Nb2C-PVP (100 μg/mL) only, and Nb2C-PVP + IR). Images share the same scale bar (100 μm). (d) TUNEL assay for determining the apoptosis of rat PBL after exposure to IR at different doses (0, 3, 6, and 9 Gy) with or without pretreatment of Nb2C-PVP (100 μg/mL). Five hundred PBL from each sample were used for TUNEL quantitation. (e) Corresponding fluorescence images of the TUNEL assay in rat PBL after different treatments. Images share the same scale bar (20 μm). (f) DCFH-DA assay showing the ROS level in 3T3/A31 cells after 6 Gy X-ray radiation with pretreatment of Nb2C-PVP at different concentrations (0, 50, and 100 μg/mL). All the values are the average from three independent experiments with the SD indicated by error bars. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the corresponding untreated control group; $$P < 0.01, $$$P < 0.001 compared with the 50 μg/mL Nb2C-PVP group at the same irradiation dose; ###P < 0.001 compared with the irradiation alone group.

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 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 Nb2CPVP 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 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 noncytotoxic Nb2C-PVP on the cell survival of 3T3/ A31 cells exposed to IR at different doses. As expected,

3c shows the CV current of Nb2C-PVP measured in H2O2 (N2-saturated). Similarly, a larger negative current density at the potential of −0.7 V was observed in the presence of Nb2CPVP compared to the unmodified GC electrode, manifesting that Nb2C-PVP has the ability toward H2O2 elimination. Therefore, the results of the CV curve imply the extraordinary catalytic abilities of Nb2C-PVP toward H2O2 and O2 reduction. It is well known that O2•− and •OH are highly destructive ROS, which can be generated by IR and induce damage in DNA, protein, and lipid in the cells.37,38 The effect of Nb2CPVP 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, SOD can inhibit the formation of formazan by eliminating O2•−. As shown in Figure 3d, when the concentration of Nb2C-PVP was 80, 100, 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 concentrationdependent way. It can be deduced that Nb2C-PVP has a SODlike activity. The •OH-scavenging activity of Nb2C-PVP was further evaluated by the electron spin resonance (ESR) measurement. The •OH was generated by the Fenton reaction 6443


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Figure 5. Radiation protection by Nb2C-PVP in vivo. (a) 30-day survival rate of BALB/C mice exposed to sublethal TBI (5 Gy) and lethal TBI (6.5 Gy) with pretreatment of 0, 5, 10, and 20 mg/kg Nb2C-PVP or 400 mg/kg AM. (b) Histological examination of mouse femur, (c) BM-MNCs count from BALB/C mice for three treatment groups (control, IR alone, 20 mg/kg Nb2C-PVP + IR) at 1, 7, and 30 days after exposure to 5 Gy TBI, (d) Representative images of MN-PCE, and (e) MN-PCE count from BALB/C mice for three treatment groups (control, IR alone, 20 mg/kg Nb2C-PVP + IR) at 1 and 7 days after exposure to 5 Gy TBI. Images share the same scale bar (10 μm). (f) CFU-S count on the splenic surfaces from BALB/C mice in three treatment groups (control, IR alone, 20 mg/kg Nb2C-PVP + IR) at 7 days after exposure to 6.5 Gy TBI. Hematological data of (g) WBC, (h) RBC, and (i) PLT from BALB/C mice in three treatment groups (control, IR alone, 20 mg/kg Nb2C-PVP + IR) at 1, 7, and 30 days after exposure to 5 Gy TBI. Nb2C-PVP (20 mg/kg) was injected intravenously 24 h before TBI. The data present the mean ± SD from 5 BALB/C mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the unirradiated control group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the IR alone group.

peripheral blood and examined PBL apoptosis after X-ray radiation with or without Nb2C-PVP pretreatment using the TdT-mediated dUTP nick end labeling (TUNEL) staining. While Nb2C-PVP treatment alone had no effects on PBL apoptosis, Nb2C-PVP pretreatment significantly decreased the TUNEL-positive cells induced by X-ray radiation (Figure 4d,e). Furthermore, Nb2C-PVP pretreatment significantly decreased the level of IR-induced ROS in 3T3/A31 cells

pretreatment 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 6444


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AM and several potent nanoradioprotectors, and a dose of 20 mg/kg Nb2C-PVP is recommended. The hematopoietic system is extremely sensitive to IR, and radiation death with sublethal 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 the hematopoietic system is characterized by bone marrow (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 BM, counts of BM 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-MNC 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. Pretreatment with Nb2C-PVP alleviated the IRinduced 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 similar to that in unirradiated normal mice. These data indicate that Nb2C-PVP accelerates hematopoietic recovery by enhancing hematopoiesis, thereby attenuating IR-induced BM damage. We further used BM-MNC count to verify the pathological change of BM cellularity post-TBI (Figure 5c). At 1 and 7 days post-TBI, BM-MNC count was significantly decreased to nearly 7% and 3%, respectively, in the IR alone mice compared to that of unirradiated control mice. In contrast, the BM-MNC count in the irradiated mice with pretreatment of Nb2C-PVP was nearly 3- and 7-fold higher than those of IR alone mice at 1 and 7 days post-TBI. At day 30, the BM-MNC count in the IR alone mice was still significantly lower than the level in unirradiated control mice, suggesting that the recovery was incomplete. In contrast, the BM-MNC count in the irradiated mice pretreated with Nb2C-PVP was much higher than that of the 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 with 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 doublestrand 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, the MNPCE 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 IRinduced depletion of BM.45 As shown in Figure 5d,e, the MNPCE frequency was significantly increased in irradiated mice, which was nearly 1.8- and 2.4-fold higher than that in unirradiated control mice at 1 and 7 days, respectively, after 5

(Figure 4f), suggesting that the radioprotective ability of Nb2C-PVP is associated with its effective free-radicalscavenging 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 pretreatment of Nb2C-PVP at 50 and 100 μg/mL for 4 or 24 h had no radioprotective effects on all the tested cancer cells. It has been reported that cerium oxide nanoparticles and 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 physiological conditions. 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 Nb2C-PVP possesses relatively high stability under physiological conditions. Next, we studied the effects of Nb2C-PVP pretreatment 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 sublethal (5 Gy) or lethal (6.5 Gy) doses.41 At a sublethal radiation dose (5 Gy TBI), the 30day survival rate was 100% in the mice with 20 mg/kg Nb2CPVP pretreatment 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 pretreatment with the median survival time of 21 days and a 95% CI of 18−25 days, 50% in the mice with 10 mg/kg Nb2CPVP pretreatment 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 pretreatment with the shortest survival time of 21 days as compared to IR alone mice with 100% mortality and a mean survival time of 15 days (95% CI: 14−16 days) (Figure 5a). For comparison, pretreatment of AM at a dose of 400 mg/kg, the highest 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 to mice in the 20 mg/kg Nb2C-PVP pretreatment 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 6445


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Figure 6. In vivo evaluation of radiation protection by Nb2C-PVP on IR-induced multiple organ degeneration. (a) Optical micrographs showing histological changes in testis, small intestine, liver, and lung of BALB/C mice from three different groups (control, IR alone, 20 mg/ kg Nb2C-PVP + IR) at 7 days after exposure of 5 Gy TBI. Nb2C-PVP was injected intravenously 24 h before TBI. All the images share the same scale bar (20 μm).

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 the Nb2C-PVP pretreatment 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. 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 an 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 the IR alone group, suggesting that a small amount of hematopoietic stem cells still survived in the body. Irradiated mice pretreated with Nb2C-PVP formed a large number of spleen nodules (11.17 ± 3.43/spleen) in the spleen, which was increased by nearly 5-fold compared to the 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 the 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 increased at 30 days postTBI. 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 the unirradiated group. In contrast, pretreatment with Nb2C-PVP significantly enhanced the WBC count, which was nearly 2- and 3-fold 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 decrease of RBC and PLT was found in mice pretreated with Nb2C-PVP at 7 days post-TBI. These data indicate that preirradiation 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 Nb2CPVP in the protection of various tissues including testis, small intestine, lung, and liver in mice irradiated with a sublethal 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 the 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-pretreated mice was restored, and the morphology was almost normal. The small intestine of irradiated mice exhibited a 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 likely resulted from retarded regeneration after IR-induced crypt epithelial cell damage. No obvious intestinal alteration was found in irradiated mice protected with Nb2C-PVP. Irradiation of lungs caused moderate pulmonary lesions. 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-PVPpretreated mice were not obvious. The examination of the liver of irradiated mice showed extensive hydropic degeneration 6446


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Figure 7. In vivo mechanism of radiation protection by Nb2C-PVP. (a) ROS levels and (b) Total SOD activities in BM-MNCs of BALB/C mice from three different groups (control, IR alone, 20 mg/kg Nb2C-PVP + IR) at 1, 7, and 30 days after exposure of 5 Gy TBI, total SOD activities, and MDA levels in (c, d) testis, (e, f) small intestine, (g, h) lung, and (i, j) liver of BALB/C mice from three different groups (control, IR alone, 20 mg/kg Nb2C-PVP + IR) at 1 and 7 days after exposure of 5 Gy TBI. Nb2C-PVP was injected intravenously 24 h before TBI. The data are presented as the mean ± SD from 5 BALB/C mice. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the unirradiated control group; #P < 0.05, ##P < 0.01 compared with the IR alone group.

dehyde (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 pretreatment on IR-induced oxidative stress by measuring the ROS level or total SOD activities and MDA content in hematopoietic tissue, testes, small intestine, lung tissues, and liver in mice exposed to sublethal TBI. As shown in Figure 7a,b, exposure of mice to a sublethal dose of TBI induced a significant increase of ROS production and a distinct decrease of total SOD activities in BM-MNCs. It was found that ROS levels in irradiated mice at 1, 7, and 30 days post-TBI were 1.74, 1.63, and 1.27 times higher than that in healthy mice,

(cellular swelling) with vacuolation and hypertrophied hepatocytes containing condensed nuclei in the hepatocytes. Nb2C-PVP pretreatment 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, and even no obvious abnormalities were observed by pretreatment with Nb2C-PVP (Figure 6). Nb2C-PVP Restores SOD Activities and Suppresses MDA Level after IR in Vivo. IR-induced ROS cause oxidative damage in cells and tissues via attacking biological macromolecules such as nucleic acids, lipids and proteins.37,38 SOD constitute the first line of defense against ROS.38 Malondial6447


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Figure 8. Pharmacokinetics, biodistribution, and metabolism of Nb2C-PVP. (a) Blood circulation time after intravenous injection of 20 mg/ kg Nb2C-PVP. (b) Biodistribution of Nb (% injected dose (ID) of Nb per gram of tissue) in main tissues after intravenous injection of 20 mg/kg Nb2C-PVP at different time intervals (4 h, 12 h, 1 d, 2 d, 7 d, and 14 d). (c) Biodistribution of Nb in BM of femurs (% ID of Nb) and BM-MNCs (pg/BM-MNC of Nb) after intravenous injection of 20 mg/kg Nb2C-PVP at different time intervals (4 h, 12 h, 1 d, 2 d, 7 d, and 14 d). (d) Accumulated Nb excretion in urine and feces after the administration of 20 mg/kg Nb2C-PVP over 7 days. Data are the average from three mice with the SD indicated with error bars.

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 halflife of 3.8 h in BALB/C mice (Figure 8a). The cysteineprotected MoS2 nanodots, an effective radioprotectant, have 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 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 nonselective 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 a 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

while total SOD activities in irradiated mice were 95%, 97%, and 46% lower than that in healthy mice, indicating that excessive ROS represses the activities of SOD, resulting in impaired ability of self-protection of mice. Compared with the IR alone group, pretreatment of irradiated mice with Nb2CPVP significantly reduced or even prevented the IR-induced production of ROS and significantly enhanced total SOD activities in BM-MNCs, showing that the level of ROS at 1, 7, and 30 days post-TBI and total SOD activities at 30 days postTBI 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 SOD 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, pretreatment with Nb2C-PVP significantly enhanced the total SOD 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 Nb2C-PVP reduces the IR-induced suppression of SOD 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 6448


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postinjection, 83.6−99.0% on day 7 postinjection, and 97.1− 99.9% on day 14 postinjection compared to 24 h postinjection upon treatment with Nb2C-PVP at 20 mg/kg (Figure 8b,c). More specifically, at 48 h postinjection, 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 postinjection, 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 postinjection, 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 feces and urine (Figure 8d) during 48 h postinjection in BALB/C mice, which is consistent with the results of tumorbearing nude mice.31 Nearly 80% of Nb could be excreted through feces (57%) and urine (23%) during 7 days postinjection (Figure 8d). Zhang et al. reported that nearly 80% of ultrasmall cysteine-protected MoS2 nanodots (50 h) and were primarily accumulated in the liver and spleen. The Bi contents in the liver (∼62 μg/g) were high after 7 days, resulting in minor liver damage after 7 days, which was recovered after 90 days when 93% Bi in the mouse body was eliminated.55 It has been reported that the Fe@C and CoNi@ C nanoshields with potent radiation protection activities possess a long blood circulation time, high liver uptake, and slow clearance due to their large particle size and surface chemistry, which could result in a potential risk of liver damage.17 The ultrathin 2D Nb2C-PVP NPs shown here possesses the unique feature of rapid clearance through the liver and kidney and little accumulation in all tissues, suggesting that Nb2C-PVP could have a favorable in vivo toxicity profile. In Vivo Toxicity of Nb2C-PVP. The biosafety of Nb2CPVP is critical for further potential clinical applications. We therefore further studied in vivo toxicity of Nb2C-PVP in healthy male BALB/C mice. The mice received intravenous injection of Nb2C-PVP at a dose of 20 mg/kg. At 7 and 30 days after intravenous injection, there were no significant changes in hematological parameters including WBC count and differential count, RBC count/size/volume, hemoglobin content, and PLT count (Figure S13), biochemical indicators of liver and kidney functions (Figure S14), and pathological changes in main organs including liver, lung, spleen, heart, kidney, testis, and small intestine (Figure S15). These findings are consistent with the results from our previous study in female Kunming mice.31 Moreover, we also evaluated the toxicity of Nb2C-PVP on BM by measuring the MN-PCE count, BM-MNC count, and pathological changes in BM. As shown in Figure S16, there were no significant differences in the MN-PCE frequency and BM-MNC count between the Nb2C-PVP-treated mice and vehicle-treated mice at 7 and 30 days after intravenous injection. In addition, the pathological changes in BM were not observed up to 30 d after intravenous injection (Figure S15). These results suggest that Nb2C-PVP NSs display excellent in vivo biocompatibility and biosafety, which is in line with the rapid clearance and almost no in vivo retention of Nb2C-PVP. The biosafety of Nb2C-PVP is comparable to the high-efficacy radioprotectant cysteineprotected MoS2 nanodots with more than 80% urinary excretion after 24 h of intraperitoneal injection in mice.22

EXPERIMENTAL SECTION Synthesis of Nb2C NSs (MXenes). The Nb2C NSs were synthesized by HF etching and TPAOH intercalating. The Nb2AlC powder (Forsman Scientific Co., Ltd. Beijing, China) was stirred in 50% HF aqueous solution (Sinopharm Chemical Reagents Co., Ltd., Shanghai, China). After 2 days, water and ethanol were repeatedly added to precipitate the Nb2C NSs and remove excess reactants combined with centrifugation at 20 000 rpm for 20 min. The precipitate was dispersed and stirred in 25% TPAOH aqueous solution (J&K Scientific Co., Ltd., Beijing, China) for 3 days at room temperature. Finally, the precipitate was repeatedly washed with water and ethanol by centrifugation, and the as-prepared raw Nb2C NSs were dispersed into the aqueous solution. Surface Modification of Nb2C NSs (Nb2C-PVP). A 4 mmol amount of Nb2C and 8 mmol of polyvinylpyrrolidone 40 (PVP40, average MW 40 000) were respectively dispersed and dissolved into 100 mL of anhydrous ethanol and refluxed at 50 °C for 6 h. The excess PVP40 was removed by centrifugation (20 000 rpm, 20 min). Afterward, the resulting Nb2C-PVP was washed with ethanol and water for further use. Characterization. TEM images and EDS spectra were obtained on a JEM-ARM300F and JEM-2100F transmission electron microscope. SEM images, STEM images, and element mapping scans were acquired on a field-emission Magellan 400 microscope (FEI Co.). XRD was measured on a Rigaku D/MAX-2200 PC XRD system (parameters: Cu Kα, λ = 1.54 Å, 40 mA, and 40 kV). AFM images were recorded by means of a Veeco DI Nanoscope Multi Mode V system. XPS was performed on an ESCAlab250 (Thermal Scientific). The N2 adsorption−desorption isotherm was tested by the Brunauer−Emmett−Teller method to characterize the specific surface area on a QuadraSorb Station 4 at 77.3 K. The quantitative analysis of elements was tested by inductively coupled plasma-optical emission spectrometry (ICP-OES) (Agilent 725, Agilent Technologies, USA). Assessment of Antioxidant Capacity of Nb2C-PVP MXenes. Antioxidant capacity assay kit with a rapid ABTS method was purchased from Beyotime Technology Inc. (S0121, China). Nb2CPVP at 0, 50, 100, 200, and 500 μg/mL in ABTS working solution was kept in the dark at room temperature for 6 min, and the absorbance at 414 nm was read by a microplate reader (Synergy H1, BioTek, USA). Test of Catalytic Activities for the Elimination of H2O2 and Oxygen of Nb2C-PVP MXenes. The three-electrode method was used to conduct the CV measurements. A carbon rod and a saturated calomel electrode were set as the counter electrode and reference electrode, respectively. Nb2C-PVP (10 μL, 1.5 mg/mL) was dropped

CONCLUSION In summary, this work has successfully demonstrated that 2D ultrathin Nb2C-PVP MXenes are highly effective in radiation protection by free radical scavenging, not only from the viewpoint of theoretical calculation but also from the systematical assessment both in vitro and in vivo. In addition, these Nb2C-PVP MXenes were featured with physiochemical properties such as high redox potential and SOD antioxidant enzyme-mimicking performance and exhibited a high efficiency of ROS scavenging with high catalytic activities against H2O2, O 2 •− , and • OH. Nb 2 C-PVP shows excellent in vivo biocompatibility and biosafety. Importantly, Nb2C-PVP significantly increased normal cell viability in vitro and mouse survival after lethal TBI. Critically, pretreatment of Nb2C-PVP 6450


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time points (15 min, 30 min, 1 h, 2 h, 4, 8 h, 24 h, and 48 h) after injection. Main organs including liver, lung, spleen, heart, kidney, testis, small intestine, and BM in femurs were harvested at different time intervals (4 h, 12 h, 24 h, 48 h, 7 d, and 14 d) after injection. The urine and feces were collected at different time intervals (12 h, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, and 7 d). In addition, BALB/C mice were intravenously injected with 5 mg/kg Nb2C-PVP (n = 3). Main organs including the liver, lung, spleen, heart, kidney, testis, small intestine, and BM in femurs were harvested at different time intervals (4, 12, 24, and 48 h) after injection. Subsequently, blood, organs, urine, and feces were treated with aqua regia. BM-MNCs isolated from femurs were counted and cell pellets were digested with alkalic solution as described in our previous report.66 The levels of Nb element were detected by ICP-OES for tissue samples at 48 h postinjection and by ICP-MS (NexION 300, PerkinElmer, USA) for BM and BM-MNC samples during 4 h to 14 days postinjection, urine and feces samples during 12 h to 7 days postinjection, and tissue samples on the 7th and 14th days postinjection. The plasma half-life of Nb2C-PVP was determined by a two-compartment pharmacokinetic model. In Vivo Survival Evaluation. In vivo radiation experiments were performed using a 137Cs γ-ray irradiator (Gammacell 40, MDS Nordion Internaional Inc., Canada) with a dose rate of 0.73 Gy/min. BALB/C mice were randomly divided into eight groups (10−16 mice in each group): (1) PBS control, (2) 5 Gy IR alone group, (3) 6.5 Gy IR alone group, (4) 20 mg/kg Nb2C-PVP pretreatment plus 5 Gy IR group, (5) 5 mg/kg Nb2C-PVP pretreatment plus 6.5 Gy IR group, (6) 10 mg/kg Nb2C-PVP pretreatment plus 6.5 Gy IR group, (7) 20 mg/kg Nb2C-PVP pretreatment plus 6.5 Gy IR group, (8) 400 mg/kg amifostine (MERRO Pharmaceutical, Dalian, China) pretreatment plus 6.5 Gy IR group. Mice were injected intravenously with Nb2CPVP in 200 μL of PBS 24 h before TBI of 5 or 6.5 Gy. All the mice were monitored for up to 30 days, and the survival of mice was recorded. Counting of BM-MNCs and Assessment of ROS Level and Total SOD Activity in BM-MNCs. BM-MNCs were isolated from femurs with a BM-MNC extraction kit (TBD2013CM) from TBDscienses Inc., China. The number of BM-MNCs was determined with a blood corpuscle counting meter under a light microscope (10× objective). The ROS level was assessed with an assay kit (S0033) from Beyotime Technology Inc., China, and the total SOD activity was measured with an assay kit (S0109) from Beyotime Technology Inc., China. MN-PCE Assay. The BM cells were flushed out of one side of the femur using RPMI-1640 medium and centrifuged at 1000 rpm for 10 min. The cell pellet was resuspended in 100 μL of calf serum, smeared on a glass slide, dried, and fixed with 100% methanol. The slides were stained with Giemsa (1:20) diluted in phosphate buffer (pH 6.8) for 20−25 min followed by washing with water. The number of MN-PCE in 1000 PCE was counted under a light microscope (100× objective) for each mouse. The frequency of MN-PCE was calculated by dividing the number of MN-PCE by the number of total PCE. Detection of CFU-S. The CFU-S was detected by endogenous spleen nodule formation. The mice were sacrificed 7 days post-TBI (6.5 Gy). The spleen was removed and fixed in Bouin’s solution (M005, Shanghai Gefan Biotechnology Co. Ltd., China) for 72 h, and the number of spleen nodules was scored in each spleen. Analysis of Hematology and Pathology. Whole blood was collected via the orbital vein, and the hematological parameters including WBC, RBC, and platelets were analyzed by a blood cell analyzer (BC-2800Vet, Mindray, China). BALB/C mice were sacrificed on days 1 and 7 post 5 Gy TBI, and femur, testis, small intestine, lung, and liver were harvested and fixed in 4% paraformaldehyde for 24−48 h, embedded in paraffin, cut into 4 μm thick sections, and stained with hematoxylin and eosin (H&E). Tissue sections were examined and photographed under a digital microscope (Imager M2, ZEISS microscope, Germany). Test of Total SOD Activity and MDA Content in Tissues. BALB/C mice were sacrificed on day 1 and 7 post-5-Gy-TBI, and livers, lungs, small intestine, and testis were harvested and homogenized in normal saline at 4 °C. After centrifugation at

and dried on a GC working electrode. The CV curves were detected on an electrochemical analyzer (CHI660D, CH Instruments, China) to evaluate the catalytic activities of Nb2C-PVP for H2O2 and oxygen elimination. Test of SOD-like Activity of Nb2C-PVP MXenes. A 20 μL amount of as-prepared Nb2C-PVP solution (80, 100, and 120 μg/mL) was mixed with the NBT/enzyme working solution, which was composed of 158 μL of SOD detection buffer, 1 μL of NBT, and 1 μL of xanthine oxidase solution from the Total SOD assay kit (S0109, Beyotime Technology Inc., China). After incubation with xanthine solution in the dark at 37 °C for 30 min, the SOD-like activity was detected at 560 nm by a microplate reader (Synergy H1, BioTek, USA). Test of •OH Scavenging Activity of Nb2C-PVP MXenes. The scavenging of •OH was detected by ESR measurement. Typically, 4 × 10−3 M H2O2 and 4 × 10−4 M FeSO4 were mixed to generate •OH. BMPO (B568-10, DoJindo Chemical Technology Inc., Japan) was used as a spin trap to identify •OH. After that, Nb2C-PVP (10, 50, and 100 μg/mL) was added into the mixture of •OH and BMPO, and ESR spectra were read by the detector (E500 10/12, Bruker, Germany). In Vitro Cytotoxicity Assay. BALB/3T3 clone A31 mouse fibroblasts (noted as 3T3/A31 cells; Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were cultured in high-glucose Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (Gibco), penicillin (100 units/mL), and streptomycin (100 mg/mL). 3T3/A31 cells were inoculated into 96well plates (3000 cells/well) and incubated under 37 °C, 5% CO2, and a humidified atmosphere for 24 h. The cells were then treated with Nb2C-PVP for 24 or 48 h. The cell viability was measured by the CCK-8 assay (40203ES60, Yeasen Biotechnology, China). Assessment of Radiation Protection in Vitro. In vitro radiation experiments were performed using an X-ray biological irradiator (XRad 320, Precision X-ray Inc., USA) at a dose rate of 2.4 Gy/min. The 3T3/A31 cells were seeded into 96-well plates at the density of 2000 cells/well. After 24 h incubation, the cells were treated with Nb2CPVP at 50 and 100 μg/mL for 4 h. HepG2, A549, and 4T1 cells were seeded into 96-well plates at the density of 2000 cells/well. After 24 h of incubation, the cells were treated with Nb2C-PVP at 50 and 100 μg/mL for 4 and 24 h. The cells were then exposed to 0, 3, 6, 9, and 12 Gy X-ray radiation. The cell viability was measured 48 h after IR using the CCK-8 assay. TUNEL Assay in Vitro. Rat peripheral blood lymphocytes were extracted according to the protocol provided by the manufacturer (LTS1083, TBDscienses Inc., China). Cell viability was tested by Trypan blue staining (TBD20180079, TBDscienses Inc., China). Cells were treated with Nb2C-PVP at 100 μg/mL in 24-well plates. After 4 h of culture, the cells were exposed to 0, 3, 6, and 9 Gy X-rays. After a further 48 h incubation, the levels of apoptosis were measured by the TUNEL FITC apoptosis detection kit (A111-03, Vazyme Biotech Co. Ltd., China). ROS Assay in Vitro. 3T3/A31 cells were seeded into 96-well plates and cultured overnight. Then, the cells were treated with Nb2CPVP at 50 and 100 μg/mL for 4 h before 4 Gy X-ray radiation. After 24 h of incubation, the cellular ROS level was measured by the ROS assay kit (S0033, Beyotime Technology Inc., China). Assessment of in Vitro Stability. Nb2C-PVP was suspended in SBF and maintained at 37 °C for varied time intervals (0, 4, 12, 24, and 48 h). The suspension was then centrifuged and washed with ethanol and water three times. The morphology and microstructure of Nb2C-PVP were systematically observed under TEM. The O and Nb element contents of Nb2C-PVP were in situ determined by EDS. Animals. The 7-week-old male BALB/C mice (20 g ± 2 g) were obtained from Shanghai Jiesijie Laboratory Animal Technology Co. Ltd. All animal experiments were performed according to the guidelines and procedures approved by the Animal Research Ethics Committee of School of Pharmacy of Fudan University. Analyses of Pharmacokinetics, Biodistribution, and Excretion. BALB/C mice were intravenously treated with 20 mg/kg Nb2C-PVP (n = 3). A 50 μL amount of blood was collected at varied 6451


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ACS Nano 12 000 rpm for 5 min at 4 °C, the supernatant was used for detecting the total SOD activity (S0109, Beyotime Technology Inc., China) and MDA content (S0131, Beyotime Technology Inc., China) according to the kit instructions. Evaluation of in Vivo Toxicity. Twelve male BALB/C mice were divided equally into two groups and treated with 20 mg/kg Nb2CPVP or vehicle (control group). Mice were intravenously injected with Nb2C-PVP in 200 μL of PBS. After 7 and 30 days, vein blood and main organs (femurs, liver, lung, spleen, heart, kidney, small intestine, and testis) were collected for hematological, blood biochemical, and pathological analyses, respectively. The BM cells from unilateral femur were used for the BM-MNC count and MNPCE assay. Statistical Analysis. All data were expressed as average ± standard deviation (SD). The data were analyzed using SPSS 17.0 software (Chicago, IL, USA). Student’s t test was used for statistical analysis between two groups. One-way analysis of variance (ANOVA) was employed to compare data among multiple groups. Survival was recorded daily for 30 days, and median survival time was evaluated by Kaplan−Meier plots. Differences in the 30-day survival rate were analyzed by the chi-square test. P values less than 0.05 were considered statistically significant.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.8b09327. SEM images, elemental mapping, XRD patterns of Nb2C NSs, preoptimized interaction site graph, XPS spectra, XRD patterns of Nb2C NSs reaction with O2•−, in vitro stability, pharmacokinetics, biodistribution, and metabolism analysis, and in vivo toxicity (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail (H.C): [email protected]. *E-mail (Y.C): [email protected]. ORCID

Han Lin: 0000-0003-1663-5468 Yu Chen: 0000-0002-8206-3325 Author Contributions

Y.C. and H.C. conceived and designed the project. X.R. synthesized and characterized the Nb2C NSs and performed in vitro and in vivo experiment. M.H. performed the DFT calculations. H.L. performed the synthesis. M.W., X.Z., and J.Y. performed the in vivo experiment. X.R., Y.C., and H.C. analyzed the data and wrote the manuscript. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We greatly acknowledge the financial support from the National Key R&D Program of China (Grant No. 2016YFA0203700), National Natural Science Foundation of China (Grant Nos. 81273000, 51722211, 51672303), and the Program of Shanghai Academic Research Leader (Grant No. 18XD1404300). REFERENCES (1) Mullenders, L.; Atkinson, M.; Bouffler, S.; Sabatier, L.; Paretzke, H. Assessing Cancer Risks of Low-Dose Radiation. Nat. Rev. Cancer 2009, 9, 596−604. (2) ICRP. ICRP Statement on Tissue Reactions/Early and Late Effects of Radiation in Normal Tissues and Organs-Threshold Doses 6452


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