Black Phosphorus Quantum Dot Induced Oxidative Stress and Toxicity

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Black Phosphorus Quantum Dots Induced Oxidative Stress and Toxicity in Living Cell and Mice Xiaoyu Mu, Junying Wang, Xueting Bai, Fujuan Xu, Haixia Liu, Jiang Yang, Yaqi Jing, Lingfang Liu, Xuhui Xue, Haitao Dai, Qiang Liu, Yuan-Ming Sun, Changlong Liu, and Xiao-Dong Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 29 May 2017 Downloaded from http://pubs.acs.org on May 29, 2017

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ACS Applied Materials & Interfaces

Black Phosphorus Quantum Dots Induced Oxidative Stress and Toxicity in Living Cell and Mice Xiaoyu Mu a, Jun-Ying Wang a, Xueting Bai a, Fujuan Xu a, Haixia Liu a, Jiang Yang b, Yaqi Jing a, Lingfang Liu a, Xuhui Xue c, Haitao Dai a, Qiang Liu c, Yuan-Ming Sun c, Changlong Liu a, ∗ and Xiao-Dong Zhang a, d,∗ a

Tianjin Key Laboratory of Low Dimensional Materials Physics and Preparing Technology,

Institute of Advanced Materials Physics, School of Sciences, Tianjin University, Tianjin, 300350, China b

Environment, Energy and Natural Resources Center, Department of Environmental Science

and Engineering, Fudan University, Shanghai, 200433, China c

Tianjin Key Laboratory of Molecular Nuclear Medicine, Institute of Radiation Medicine,

Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, 300192, China d

Tianjin Collaborative Innovation Center of Chemical Science and Engineering, Tianjin,

300072, China

Keywords: Black Phosphorus Quantum Dots; Toxicity; Oxidative stress; Reactive oxygen species; Stability

∗ Correspondence should be addressed to X.Z. ([email protected]), C.L. ([email protected]) 1

ACS Paragon Plus Environment


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Abstract: Black phosphorus (BP), as an emerging successor to layered two-dimensional materials, has attracted extensive interests in cancer therapy. Toxicological studies on BP are of great importance for potential biomedical applications, yet not systemically explored. Herein, toxicity and oxidative stress of BP quantum dots (BPQDs) at cellular, tissue and whole-body levels are evaluated by performing the systemic in vivo and in vitro experiments. In vitro investigations show that BPQDs at high concentration (200 µg/mL) exhibit significant apoptotic effects on HeLa cells. In vivo investigations indicate that oxidative stress, including lipid peroxidation, reduction of catalase activity, DNA breaks and bone marrow nucleated cells (BMNC) damage, can be induced by BPQDs transiently but recovered gradually to healthy levels. No apparent pathological damages are observed in all organs, especially in the spleen and kidneys, during the 30-day period. This work clearly shows that BPQDs can cause acute toxicities by oxidative stress responses, but the inflammatory reactions can be recovered gradually with time for up to 30 days. Thus BPQDs do not give rise to long-term appreciable toxicological responses.

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1. Introduction As a novel family of nanomaterials, layered two-dimensional (2D) materials, including grapheme, boron nitride, Bi2Se3, and transition-metal dichalcogenides (TMDs), have attracted extensive attention on both fundament research and practical applications due to their unique physical, chemical, and electronic properties1-7. Moreover, originating from quantum confinement and edge effects8-9, quantum dots (QDs) with ultra-small size and ultrathin thickness exhibit some distinctive properties, enabling them promising in applications for photovoltaics10, optoelectronic devices11, and biomedicine3-7. Recently, black phosphorus (BP) nanomaterials with different thicknesses and sizes have been successfully prepared via mechanical exfoliation or liquid-phase ultrasonication techniques12-20. As a successor to 2D layered materials, BP nanomaterials attracted broad research interests in many fields, such as composites and sensors14,

21

, field-effect

transistors22-27, photodetectors28-29, thin-film solar cells30-31, and anode materials in lithium-ion batteries32-33. BP is not only the most stable allotrope of elemental phosphorus, but also a semiconductor with a direct and tunable band gap varying from about 0.3 eV in bulk form to ~2.0 eV in monolayer form34-36. Thus, BP shows broad absorption across the UV and IR regions. Furthermore, phosphorus is one of the vital elements in organism and thus BP exhibits innate biocompatibility37-39, indicating the potential biomedical applications of BP nanomaterials. For instance, ultrathin BP nanosheets could efficiently generate singlet oxygen as photodynamic therapy (PDT) agents16. Moreover, BPQDs and BP nanoparticles (NPs) are promising for photothermal therapy (PTT) of cancer40-42, due to the high near infrared (NIR) extinction coefficient and photo-thermal conversion efficiency. It is noteworthy that the theme 3



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of reactive oxygen species (ROS) generation and oxidative stress responses not only has become an established paradigm to assess NPs toxicity7, 43-52, but also is closely related to the aforementioned cancer therapies16, 53-58. Thus, it is interesting to examine oxidative stress responses induced by BPQDs and further reveal the biointerface interaction as well as nanotoxicology. However, the biomedical studies on BP nanomaterials are still in early stages and systematic investigations on the toxicity of BPQDs in vitro and vivo are scarce. In this work, the toxicities of BPQDs at cellular, tissue and whole-body levels have been systematically evaluated by a series of in vitro and in vivo experiments. Apoptosis staining using Annexin V and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) suggest mild toxicity to HeLa cells and liver tissues 24 h after injection of BPQDs, ascribed to oxidative stress such as ROS induced by BPQDs. The body weight of mice has also decreased after injection of BPQDs, indicating acute toxicity. Surprisingly, the oxidative stress on liver, DNA and bone marrow nucleated cells (BMNC) undergoes a self-repair mechanism and body weight can gradually recover to healthy levels over time. Hematological, biochemical and pathological analyses demonstrate that BPQDs do not induce significant long-term infection or inflammation in mice.

2. Experimental Section Preparation of black phosphorous quantum dots: The BP bulk crystals were purchased from a commercial supplier (99.998 %, Nanjing XFNANO Materials Tech Co., Ltd) and stored in a N2 glove box prior to use. The preparation of BPQDs is given in detail as follow. 1.5 g of BP bulk crystal was added in a mortar in batches (150 mg per batch) and then 4


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grounded by hand for 2 hours per batch in a N2 glove box. For fully grinding down into micron-order flour, small amount of NMP (99.5 %, anhydrous, Aladdin Reagents) was frequently added into the mortar and the supernatant mixture was timely transferred to a 200 mL round-bottom flask. Finally, the mixture contained 150 mL NMP. When the flask was hermetically sealed, it was fetched out from the glove box. Then, the flask containing BP mixture in an ice bath was treated by ultrasonic continuously for 15 h at the power of 200 W. The resulting solution was centrifuged at 7,000 rpm for 20 min and the supernatant solution containing BPQDs was collected to centrifuge at 12,000 rpm for another 20 min. The precipitates were repeatedly washed with deionized water for at least three times to entirely remove the initial solvent and re-suspended in water. Thus, the BPQDs aqueous solution was prepared. Characterization: UV-vis absorption spectra were recorded at room temperature on a Shimadzu UV-3600 double-beam spectrophotometer with QS-grade quartz cuvettes. Raman spectrum was performed at room temperature on a micro-Raman spectrometer with a 532 nm excitation wavelength and 1800 lines mm-1 grating (Renishaw INVIA Reflex). Raman band of the spectrometer was calibrated using a silicon wafer at 520 cm-1. In addition, in situ Raman spectra were conducted with a 785 nm excitation wavelength and 1200 lines mm-1 grating to evaluate the stability of BPQDs. Fourier transform infrared (FTIR) spectroscopy was performed on a Perkin Elmer Frontier spectrometer and samples were prepared in KBr pellets. To characterize the morphology of BPQDs, high-resolution transmission electron microscopy (HRTEM) analysis was taken on the JEOL JEM-2100F transmission electron microscope with an acceleration voltage of 200 kV. For atomic force microscopy (AFM) 5



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analysis, the BPQDs dilute solution was dropped onto a preheated (80 ºC) SiO2/Si wafer that was serially washed by sonication in acetone, ethanol and deionized water, respectively. The AFM images of BPQDs were then recorded in tapping mode in air using a Multi Mode 8 microscope (Bruker) and processed by NanoScope Analysis software. Cell apoptosis assay: To detect apoptotic and necrotic cells, FITC Annexin V Apoptosis Detection Kit (BD company, US) was employed. The instructions of manufacturer were strictly followed. Briefly, HeLa cells in the 37 ºC incubator containing 5 % CO2 were incubated for 72 h. After the BPQDs with 0, 50, 100 or 200 µg/mL were introduced to the cells, they were incubated for another 24 h. Then, the cells were bathed twice with phosphate-buffered saline (PBS) and resuspended in the buffer solution to 1 × 106 cells/mL. The above-mentioned cells solution (100 µL), annexin V-FITC (5 µL) and propidium iodide (PI, 5 µL) were successively added to a tube. After incubation for 15 min in dark at room temperature, 400 µL PBS was added to dilute the stained HeLa cells and fluorescence-activated cell sorting (FACS) was applied to analyze the cell apoptotic and necrotic. Intracellular

reactive

oxygen

species

assay:

The

oxidant-sensitive

dye

(2,7-dichlordihydrofluorescein diacetate, DCFH-DA) was used for ROS detection. Briefly, HeLa cells placed in the 6-well plates (1 × 104 cells per well) were incubated for 24 h. After the BPQDs with 0, 50, 100 and 200 µg/mL were added to the cells, they were incubated for another 24 h. Next, the fresh culture medium, including 5 µM DCFH-DA (1 mL) was employed to replace the culture medium for all plates. After incubation for 20 min in dark at room temperature, the cells were bathed with PBS for three times and then observed by 6


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fluorescence microscope. In vitro DNA damage quantification: The evaluation of DNA damage was performed by a revised protocol of comet assay. Agarose (0.8%, 500 µL) was coated homogeneously on glass microscopic slides. After solidification, 4.8 × 104 cells in 30 µL PBS incubated with BPQDs (200 µg/mL) were mixed with the agarose (0.6%, 70 µL), and 20 µL of this mixture was spread over the slide completely. The solidified slides were placed into the newly prepared ice lysis buffer solution (1% Triton X-100, 10% DMSO, 100 mmol/L Na2EDTA, 10 mmol/L Tris-HCl, and 2.5 mol/L NaCl) for 2 h, and then immersed into a horizontal gel electrophoresis unit that was filled with chilled electrophoresis buffer (pH 7.4, 300 mmol/L NaOH, 1 mmol/L Na2EDTA) for 30 min. After electrophoresis for 20 min at 30 V, the slides were neutralized with ethyl alcohol and stained with ethidium bromide (2 µg/mL). Finally, the Comet Assay Software Project was adopted to analyze the tail DNA and tail moments by counting 100 cells. In vivo toxicity: 48 male C57BL/6 mice were purchased, fed, and processed based on the protocols approved by the Institute of Radiation Medicine, Chinese Academy of Medical Sciences (IRM, CAMS). The mice were randomly divided into 6 groups: 1-day control group, 1-day treatment group, 7-day control, 7-day treatment group, 30-day control, and 30-day treatment group respectively. 200 µL of the BPQDs solution (1.7 mg/mL) was injected into the mice via peritoneal cavity. Body weight was recorded and the behavioral changes of mice were assessed every day. At 1, 7 and 30 days, mice were killed by exsanguinations and the blood was collected to detect the hematological and biochemical indexes. During the autopsy, heart, liver, spleen, lung, kidneys, testis, brain, bladder, thymus 7



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and fur were collected and weighed for pathological and biodistribution analyses. Superoxide dismutase (SOD), 3,4-methylenedioxyamphetamine (MDA) and catalase (CAT) levels of liver were measured by UV-vis spectroscopy. Meanwhile, bilateral femurs of each mouse were excised from the body and connective tissues were removed completely to monitor DNA level and BMNC numbers. SOD, MDA and CAT levels analyses: At different time points, the apex (about 0.2 g) of the above-collected livers were cut off and placed into 10 mL centrifuge tubes. PBS was added to make 10 % tissue homogenates by a tissue homogenate machine (IKA, T18 basic). The standard operating procedures of SOD, MDA and CAT levels analyses were taken according to the directions by Superoxide Dismutase assay kits, Malondialdehyde assay kits and Catalase assay kits (Nanjing Jiancheng Bioengineering Institute). Immunofluorescence assay: As collected above, the livers of BPQDs-treated mice at 1, 7 and 30 days were steeped in 10 % formalin and then embedded in paraffin to slice. Paraffin-embedded 5 µm liver sections were obtained for immunofluorescence staining using a TUNEL kit (Beyotime, C1090). Bone marrow DNA and BMNC evaluations: To estimate the total DNA levels in bone marrows, bone marrow cells were stabbed and douched from the femurs into 10 mL 5 mM calcium chloride solution with a 24-gauge needle and as such, single-cell suspensions were prepared. After the suspensions were placed for 30 min at 4 ºC, they were centrifuged for 15 min at 2,500 rpm with supernatants discarded. The precipitate were mixed with 5 mL 0.2 M perchlorate and placed in 90 ºC water bath for 15 min. After being cooled down to the room temperature, the mixtures were purified through filter paper (pore size =0.2 ߤm) and the 8


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filtrates were measured for UV-vis absorption at 268 nm. Similarly, BMNC were flushed into 1 mL PBS, and then filtered through nylon meshes to remove fragments of the bones and tissues for flow cytometric analysis (Mindray, BC-2800vet). Hematology, biochemistry and pathology: Following the normal blood collection procedures, part of the drawn blood (20 µL) was put into potassium EDTA collection tubes for hematological analysis. The remaining blood (about 1 mL) were placed in a 4 ºC refrigerator overnight and then centrifuged for 5 min at 6,000 rpm to separate serum for biochemical examination. For pathology examination, the above-collected major organs were routinely fixed in 10 % formalin, embedded into paraffin, sliced, stained by hematoxylin and eosin (H&E), and observed using a digital microscope. Biodistribution: As collected above, the organs of BPQDs-treated mice were digested using a microwave system (CEM Mars 5, Kamp Lintfort, Germany). The phosphorus content was measured by an inductively coupled plasma mass spectrometer (ICP-MS, Agilent 7500 CE, Agilent Technologies, Waldbronn, Germany). Statistical analysis: All of the data were expressed as mean ± standard deviations (SD). Each test was repeated three or more times. Analysis of variance (ANOVA) statistics was performed, and p -values less than 0.05 were considered to be statistically significant.

3. Results and Discussion 3.1 Morphology and characterization. A liquid ultrasonication exfoliation method was adopted to prepare the BPQDs12. Figure 1a shows absorption spectrum of the BPQDs in water with a wide absorption band across the 9



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UV and NIR areas, consistent with previous reports14, 40, 42. Furthermore, as shown in Figure 1b, Raman spectrum of BPQDs exhibits three obvious peaks at 361.1 cm-1, 438.3 and 465.6 cm-1, which can be attributed to one out-of-plane phonon mode (A1g) and two in-plane modes (B2g and A2g), respectively, further confirming that the crystalline feature of BPQDs still remain after exfoliation from bulk BP crystal17, 27. Especially, the modes of A1g , B2g and A2g have the same trend of blue shifts toward higher wavenumbers compared to bulk BP, which is mainly ascribed to the decrease in thickness and transverse dimensions12,

36

. This

hypsochromic effect is similar to the shift phenomenon observed in BN QDs59 and MoS2 QDs60. The morphology of as-prepared BPQDs was characterized by TEM and AFM images, as shown in Figure 1c and 1f. The statistical result exhibits that BPQDs have an average size of 2.7±0.7 nm (Figure 1e). HRTEM image of BPQDs reveals lattice fringes of 0.34 nm in Figure 1d, corresponding to the (021) plane of BP crystal. The height profiles in Figure 1g are representative of the AFM image in Figure 1f and show that BPQDs possess a thickness ranging from ~ 0.6-2.3 nm, corresponding to 1-4 layers. Statistical analysis on AFM observations (Figure 1h) demonstrates the average thickness is 1.5±0.8 nm. 3.2 Stability evaluation under different conditions. To evaluate the stability, BPQDs (6.4 ppm.) were dispersed in water and placed in ambient air in dark for 5 days. Their absorption spectra were measured at fixed time intervals, as shown in Figure 2a. The inset photographs in Figure 2c show that although the color of the BPQDs solution becomes slightly lighter after 5 days, the Tyndall effect still exists (inset in Figure 2a) and no obvious aggregation or sedimentation is found, indicating the dispersion 10


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is still stable. Nevertheless, the BPQDs aqueous solution reveals a decrease in the overall absorption intensity over time. Figure 2c shows the variation in the absorbance ratio (A/A0) at 256 nm decreases by ~9.7% after 1 day and 34.0% after 5 days. In the first two days, the relationship between the absorption ratio and time is approximately linear with a slope of ~0.4 %·h-1. To clarify the stability of BPQDs under different conditions, in situ Raman spectra of BPQDs after 785 nm-laser irradiation at different time points were measured (Figure 2b). The inset in Figure 2b shows the magnified peak ascribed to out-of-plane phonon mode (A1g ) and its intensity decreases along with a blue shift in peak frequency as irradiated time increases. The variation in the intensity ratio at A1g is given in Figure 2d and the intensity (I) decreases by ~7.3% compared with the original value (I0) after 15 minutes. Moreover, the slope is ~27.9 %·h-1, much larger than that under dark ambient, indicating their poor stability under light exposure. In addition, FTIR spectrum of BPQDs in water after 3 days of storage displays two broad absorption bands centered at ~1000 and ~1200 cm-1, which can be attributed to the P-O stretching and P-P-O linear stretching modes61, respectively, indicating that the BPQDs in water were oxidized partially. In aqueous environment, BPQDs irreversibly react with oxygen and water to form phosphoric oxides (PxOy), followed by conversion of PxOy into PO43- anions62. Hence, the decrease in absorbance and Raman intensity is due to degradation of the BPQDs, which further highlights their promising applications in biological areas, as they can be readily degraded into biocompatible ions. 3.3 In vitro cell apoptosis and oxidative stress on HeLa cells. Annexin V-FITC/PI staining was applied to analyze dose-dependent effect of BPQDs on 11



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cell apoptosis. As shown in Figures 3a and 3c, the percentage of living cells decreases significantly with an increase of the BPQDs concentrations from 50 to 200 µg/mL. After incubation with 200 µg/mL BPQDs for 24 h, the percentage of viable cells is 36.6 %, which is half less than the control group without treatment of BPQDs, indicating that BPQDs at high concentration have significant effects on the cell viability and induce significant cell apoptosis and necrosis. To explore the reason for HeLa cell apoptosis and necrosis, endocellular ROS levels were detected utilizing a cell permeable fluorescent dye (DCFH-DA) after incubation with BPQDs at different concentrations. As shown in Figure 3b, the control group without BPQDs exhibits minimal DCF fluorescence, while strong fluorescence is observed in cells after incubation with BPQDs for 24 h at 37 ºC with 5 % CO2. Moreover, the fluorescence intensity is gradually enhanced with the increasing concentrations of BPQDs in Figure 3d, indicating generation of ROS inside the cells treated with high concentrations of BPQDs. Zhang and Xie et al reported that ultrathin BP nanosheets can generate singlet oxygen under visible light irradiation16. Although the cells were mostly kept in dark during incubation expect during handling, the entire preparation of BPQDs was almost exposed to the natural light. The duration of natural light exposure was estimated for ~17 h. Therefore, ROS, such as singlet oxygen, may be generated under room light irradiation. In addition, the stability evaluation of BPQDs under dark ambient conditions and 785 nm-laser irritations showed that BPQDs can be oxidized by water and oxygen either under dark or light irritation and the light irritation can promote the oxidation rate. The oxidation process indicated that there were electron transfer reactions between BPQDs and ground state oxygen, which could be the sources of ROS in Figure 3. These free radicals are able to react with intracellular 12


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lipids, proteins, DNA and RNA, causing structural and functional changes and affecting biological processes63-64. The DNA damage was evaluated by comet assays on CHO cells treated with or without BPQDs (200 µg/mL) and the corresponding quantitative investigations of tail DNA and tail moment are given in Figure S2. No obvious cell tail DNA and tail moment are found in the control group, while significant damages are observed in the treated group, suggesting easily identifiable DNA damages after treatment with the BPQDs. As a result, free radical damages can induce cell apoptosis and further inhibit cell proliferation. 3.4 In vivo cell apoptosis and oxidative stress on liver. To further evaluate the toxicity of BPQDs on tissues, SOD, CAT, and MDA analyses were employed to provide some insights. Generation of ROS can adversely alter lipids and proteins. Polyunsaturated fatty acids (PUFA) in biological membranes are particularly vulnerable to attack from free radicals and lipid peroxidation is caused that can give rise to infaust effects. MDA, as the product from lipid peroxidation, can induce functional and metabolic disorders or even cell death. Therefore, the amount of MDA usually can reflect the level of lipid peroxidation in vivo and indirectly disclose the degree of cell damage. Figure 4b shows the MDA content of liver from mice with or without treatments of BPQDs. The MDA content in the liver of normal mice is 0.7 nmol/mgprot, but sharply increases to 1.5 nmol/mgprot for mice treatment with of BPQDs after 1 day. After 7 days, the MDA content decreases to 0.6 nmol/mgprot in liver from mice treated with BPQDs and almost recovers to normal levels. After 30 days, MDA remains at normal levels for the treated mice (Figure 4b). MDA is normally present at low level in the body. A large quantity of MDA is produced for 13



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mice injected with BPQDs after 24 hours, indicating that ROS are generated in liver. In addition, enzyme activities can be decreased as a result of free radical damages to protein. Cells are endowed with protective antioxidant elements against adverse effects of free radicals, such as SOD, CAT, glutathione, glutathione peroxidase, glutathione reductase etc. -

SOD can catalyze the dismutation of superoxide (O2 ) radicals to either ordinary molecular oxygen (O2) or hydrogen peroxide (H2O2) and CAT can break H2O2 down to water65-66. Figures 4a and 4c show the activities of SOD and CAT in liver, respectively. The SOD activity has no significant difference in liver from mice with or without treatment of BPQDs over 30 days (Figure 4a). Nevertheless, the CAT activity in the liver of normal mice is 40.0 U/gprot, but it sharply decreases to 23.1 U/gprot for mice with treatment of BPQDs after 1 day (Figure 4c). Similar to the change in MDA content, the CAT activity exhibits powerful recovery after 7 days and is well maintained at a value close to normal level after 30 days for treated mice, clearly elucidating the ability of ROS clearance. To directly observe the free radical damage in liver after treatment of BPQDs, immunofluorescence staining was performed by the TUNEL method, as shown in Figure 4d. Living cells are stained blue and TUNEL-positive apoptotic cells are stained red. 1 day after injection, a few apoptotic cells are observed in the liver sections. Liver cells become viable and apoptotic cells are almost absent after 7 days and 30 days, illustrating that BPQDs have no long-term toxicity. It is clear that when BPQDs are injected to the mice body, lipid peroxidation and reduction of catalase activity occur in liver after 1 day, but these adverse effects can be recovered to the normal levels after a week without recurrence in a month. 3.5 In vivo toxicity. 14


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ROS can result in oxidative damage to DNA as well by adding to basics or abstracting hydrogen atoms away from the sugar moiety63. Total DNA were collected from bone marrow cells. Figure 5a gives an assessment of DNA damage for mice treated with or without BPQDs. DNA from healthy mice shows an optical density (OD) of 0.08, but it decreases to 0.05 1 day after treatment with BPQDs, indicating DNA damage to some extent induced by injection of BPQDs. After 7 days, OD of total DNA for mice treated with BPQDs recovers to 0.06, closer to the healthy value. After 30 days, OD from treated mice is stably maintained at 0.06, presenting distinct recoveries and effective DNA repairs. The number of BMNC is presented in Figure 5b. Similar to the results of DNA damage, the BMNC number decreases from 21.3 × 106 cells/mL in healthy mice to 16.4 × 106 cells/mL in treated mice after 1 day, and recovers to 21.4 × 106 and 20.8 × 106 cells/mL in treated mice after 7 and 30 days, respectively. The results of Figures 5a and 5b clearly illustrate that BPQDs do not cause long-term damages on DNA and BMNC. Meanwhile, in vivo toxicities of BPQDs are evaluated in terms of hematological and biochemical panels at 1, 7 and 30 days. Standard hematological biomarkers were examined, including white blood cells (WBC), red blood cells (RBC), hematocrit (HCT), hemoglobin (HGB), platelets (PLT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC). Hematological results from the mice injected with BPQDs are presented in Figures 5c, 5d and Figure S3. Compared the BPQDs-treated mice with untreated mice, WBC and RBC, as two representative indexes do not reveal any statistical differences (Figures 5c and 5d), clearly indicating that no significant inflammation and infection was induced by the BPQDs in mice 15



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and are relatively safe in preclinical settings. In addition, the standard biochemistry examination is performed on the mice treated with BPQDs at 1, 7 and 30 days, as shown in Figures 5(e – i) and Figure S4. The biochemical indices containing aspartate aminotransferase (AST), alanine aminotransferase (ALT), serum creatinine (CERA), glucose (GLU), globulin (GLOB), albumin (ALB), blood urine nitrogen (BUN) and total protein (TP) were investigated. Here, since ALT, AST and CREA have a close correlation to the functions of the liver and kidneys of mice, they are mainly studied. All ALT, AST and CREA do not show statistical differences between untreated and treated mice (Figures 5e, 5f and 5g). However, one can see from Figure 5h that GLU sharply decreases from 3.4 mmol/L in healthy mice to 1.4 mmol/L in treated mice after 1 day. At the middle time point (7 days), GLU is still at low levels without distinct recoveries, while all other indicators start to display appreciable recoveries to normal levels (Figure 5i and Figure S4). After 30 days, all the biochemical parameters of mice treated with BPQDs are thoroughly restored to normal. Therefore, the results explain that BPQDs do not give rise to long-term inflammatory responses, liver and kidney toxicities. 3.6 Weight, excretion, biodistribution and pathological evaluation. Body weight and behaviors of the treated mice were monitored to evaluate in vivo toxicities of BPQDs on a daily basis. Treatment with BPQDs does not induce any visible adverse reaction on the behaviors of mice during the 30 days, while body weight of the treated mice decreases slightly after 1 day (Figure 6a). After that, the body weight of mice starts to recover gradually till the 14th day to control levels. For further medical applications, pharmacokinetics and biosafety are the two most important characteristics to be taken into 16


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consideration. Male C57BL/6 mice were injected with 1.7 mg/mL BPQDs solution. Blood, urine and feces were collected for pharmacokinetic and renal-excretion studies, as measured by ICP-MS. Figure S5 shows that half-life of the BPQDs in the blood is 0.8 h. Furthermore, the majority of BPQDs can be rapidly excreted through the urine and feces route 24 h after injection due to their ultrasmall size67 (Figure 6b). To explore the final format of BPQDs after their excretion from mice, Raman spectra were measured as shown in Figure S6. Generally, BPQDs show three prominent Raman peaks at 361.1 (A1g ), 438.3 (B2g) and 465.6 (A2g ) cm-1, respectively. However, no characteristic peaks of BPQDs were observed in the Raman spectra from urine and feces, indicating that the final format of BPQDs after its excretion may not be the original BPQDs or beyond the detetino threshold. For the urine (Figure S6a), two prominent peaks can be observed at 548 cm-1 and 1013 cm-1, ascribed to the asymmetric deformation of PO4 units68 and the υ1 and υ3 vibrations of PO4 units69, respectively. For the feces (Figure S6b), only a small peak could be corresponding to the symmetric deformation of PO4 units at 400 cm-1 68. Although the excretion from blank mince contains phosphate and phosphite, we surmise that the final format of BPQDs after its excretion is PO43- species, in agreement with the oxidation of the BPQDs solution in air. For biodistribution analysis, the concentrations of P in major organs of mice with BPQDs injection were measured by ICP-MS after 1 and 30 days (Figure 6c). Finally, the pathological figures of major organs are demonstrated by histology at 1, 7 and 30 days. Heart, liver, spleen, lung, and kidneys were collected, sliced and stained by H&E. As shown in Figure 6d, no apparent damages were detected in all organs, especially in the liver, spleen and kidneys, during the entire time periods. 17



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4. Conclusion In summary, the toxicity and oxidative stress of BPQDs at cellular, tissue and whole-body levels were evaluated by performing the systemic experiments in vitro and in vivo. In vitro cell assays show that BPQDs can generate an appreciable amount of ROS resulting in cell apoptosis. In vivo experiments indicate that oxidative stress, including lipid peroxidation, reduction of catalase activity, DNA breaks and decrease of BMNC numbers, can be induced by BPQDs transiently but recovered gradually to normal levels. Hematological, biochemical and pathological analyses demonstrate that BPQDs do not give rise to the long-term inflammatory reactions and obvious damages in mice.

Supporting Information Figure S1. FTIR spectra of BPQDs dispersed in water and NMP after 3 days. Figure S2. Evaluation of DNA damage of BPQDs. (a, b) In vitro images of comet assays on CHO cells treated with or without BPQDs (200 µg/mL). (c) Tail DNA and (d) tail moment analysis. * p < 0.05 as compared with the control group (Student’s t test). Figure S3. Hematological data of mice treated with or without BPQDs at 1, 7 and 30 days including mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), hemoglobin (HGB), platelets (PLT), and hematocrit (HCT). * p < 0.05 as compared with the control group (Student’s t test).

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Figure S4. Blood biochemistry analysis of mice treated with or without BPQDs at 1, 7 and 30 days including albumin (ALB), blood urea nitrogen (BUN) and total protein (TP). * p < 0.05 as compared with the control group (Student’s t test). Figure S5. Time-dependent concentrations of P in the blood at different time points after injection. The Supporting Information is available free of charge on the ACS Publications website.

Acknowledgements This research was supported by the National Natural Science Foundation of China (Nos. 81471786, 81000668), the Natural Science Foundation of Tianjin (No. 13JCQNJC13500), the Foundation of ‘Peiyang Young Researcher’ Program of Tianjin University.

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Figure 1. Characterization of BPQDs. (a) UV/Vis absorption spectrum of the BPQDs aqueous solution. (b) Raman spectrum of BPQDs recorded using a 532 nm laser. (c) TEM image of BPQDs. (d) HRTEM image of BPQDs. (e) Statistical analysis of the sizes of BPQDs measured by TEM. (f) AFM image of BPQDs. (g) Height profiles along the white lines in (f). (h) Statistical analysis of the heights of BPQDs measured by AFM.

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Figure 2. Stability evaluation under different conditions. (a) Absorption spectra of the BPQDs (6.4 ppm) in water after storage in dark ambient conditions for different periods of time. Inset in (a): Tyndall effect. (b) In situ Raman spectra of BPQDs after 785 nm-laser irradiation of different time periods. (c) Variation of the absorption ratios (A/A0) at 256 nm. Insets in (c): Photographs of the BPQDs after storing in water for 0 and 5 days. (d) Variation of the intensity ratios (I/I0) at the Raman peak of one out-of-plane phonon mode (A1g ).

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Figure 3. Cell apoptosis and oxidative stress on HeLa cells. (a) Cell apoptosis dependent on the concentration of BPQDs. Viable cells are shown in the lower left quadrant, early apoptotic cells are shown in the lower right quadrant, and necrotic (or late apoptotic) cells are shown in the upper right quadrant. The corresponding percentages of viable, apoptotic and necrotic cells are presented in graph c. (b) intracellular ROS production in cells treated with the different concentration (0, 50, 100 and 200 µg/mL) of BPQDs. The corresponding analysis on fluorescence intensity of ROS level images is given in graph d.

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Figure 4. In vivo oxidative stress and cell apoptosis on liver. (a) SOD, (b) MDA and (c) CAT levels in liver after treatments of BPQDs at 1, 7 and 30 days. * indicates p < 0.05 as compared with the control group (Student’s t-test). (d) TUNEL analysis of liver after treatments of BPQDs at 1, 7 and 30 days. TUNEL-positive apoptotic cells are stained red.

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Figure 5. In vivo toxicity. (a) DNA damage of mice after treatment with BPQDs, as measured by UV−vis absorption at 268 nm. (b) Counts of bone marrow nucleated cells (BMNC) in mice with BPQDs injection. (c) and (d) Hematological data of WBC and RBC in the BPQDs-treated mice, respectively. (e) - (i) Blood biochemistry analysis of the BPQDs-treated mice. The indicators include ALT, AST, CREA, GLU and GLOB. All Data were collected at 1, 7, and 30 days after intraperitoneal injection. * indicates p < 0.05 as compared with the control group (Student’s t test).

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Figure 6. Weight, excretion, biodistribution and pathological evaluation. (a) Body weight of the BPQDs-treated mice during 30 days. (b) Cumulative excretion of urine and feces at different time points. (c) Biodistribution of P in major organs of mice without or with BPQDs injection at different time points post-injection. (d) Pathological evaluation (haematoxylin 34


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and eosin stained images) obtained from the liver, spleen, kidney, heart and lung of the BPQDs-treated mice at 1, 7 and 30 days post-injection.

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Black Phosphorus Quantum Dot Induced Oxidative Stress and Toxicity

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