size effect on the cytotoxicity of layered black phosphorus...

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Size Effect on the Cytotoxicity of Layered Black Phosphorus and Underlying Mechanisms

Xuejiao Zhang, Ziming Zhang, Siyu Zhang, Dengyu Li, Wei Ma, ChuanXin Ma, Fengchang Wu, Qing Zhao,* Qingfeng Yan,* and Baoshan Xing*

1. Introduction

Over the last few years, 2D crystals, such as graphene, boron nitride, and transition metal dichalcogenides, have received considerable attentions due to their outstanding performance

DOI: 10.1002/smll.201701210

A systematic cytotoxicity study of layered black phosphorus (BP) is urgently needed before moving forward to its potential biomedical applications. Herein, bulk BP crystals are synthesized and exfoliated into layered BP with different lateral size and thickness. The cytotoxicity of as-exfoliated layered BP is evaluated by a label-free real-time cell analysis technique, displaying a concentration-, size-, and cell type-dependent response. The IC50 values can vary by 40 and 30 times among the BP sizes and cell types, respectively. BP-1 with the largest lateral size and thickness has the highest cytotoxicity; whereas the smallest BP-3 only shows moderate toxicity. The sensitivity of three tested cell lines follows the sequence of 293T > NIH 3T3 > HCoEpiC. Two possible mechanisms for BP to induce cytotoxicity are proposed and verified: (1) the generation of intracellular reactive oxygen species (ROS) is detected by a ROS sensitive probe using the inverted fluorescence microscopy and flow cytometry; (2) the interaction of layered BP and model cell membrane is examined by quartz crystal microbalance with dissipation, illustrating the disruption of cell membrane integrity especially by the largest BP-1. This systematic study of BP’s cytotoxicity will shed light on its future biomedical and environmental applications.

2D Materials

Prof. X. Zhang, Prof. S. Zhang, D. Li, W. Ma, Prof. Q. ZhaoKey Laboratory of Pollution Ecology and Environmental EngineeringInstitute of Applied EcologyChinese Academy of SciencesShenyang 110016, ChinaE-mail: [email protected]

Z. Zhang, Prof. Q. YanDepartment of ChemistryTsinghua UniversityBeijing 100084, ChinaE-mail: [email protected]

D. Li, W. MaUniversity of Chinese Academy of SciencesBeijing 100049, China

Dr. C. Ma, Prof. B. XingStockbridge School of AgricultureUniversity of MassachusettsAmherst, MA 01003, USAE-mail: [email protected]

Dr. C. MaDepartment of Analytical ChemistryThe Connecticut Agricultural Experiment StationNew Haven, CT 06504, USA

Prof. F. WuState Key Laboratory of Environmental Criteria and Risk AssessmentChinese Research Academy of Environmental SciencesBeijing 100012, China

in various areas ranging from electronics, photo nics, bio-sensor, and diagnostics/therapeutics.[1] Likewise, as a newly emerging 2D material, layered black phosphorus (BP) has attracted enormous interests since 2014.[2] Owing to its unique optical and electrical properties, it is promising for

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many applications especially in the biomedical field.[3] A number of in vitro and in vivo studies have suggested layered BP a promising candidate for photothermal/photodynamic therapy in treating a variety of cancers, including breast cancer,[3b,c,4] cervical cancer,[5] and glioma.[3b] In addition, it is considered as an efficient drug delivery vehicle for syner-gistic cancer therapy.[4,5] The above-mentioned applications will inevitably make layered BP exposure to human. Hence, an in-depth understanding of BP’s cytotoxicity especially to human is of utmost importance.

While BP derivatives and nanodots (100 nm) was only studied in the concentration range from 0.025 to 0.2 µg mL−1 which were also nontoxic.[3c] However, Latiff et al. demonstrated that BP’s cytotoxi-city was generally intermediate between that of graphene oxide (GO) and exfoliated transition-metal dichalcogenides without clarifying the size of BP.[6] The controversy may be, on one hand, due to the size effect, which has already been observed for graphene and its derivatives. More than 80% viability of A549 cells were preserved by the treatment of large (780 ± 410 nm) and middle size (430 ± 300 nm) GO at the concentration of 200 µg mL−1, whereas small GO (160 ± 90 nm) decreased the cell viability to 67% after 24 h exposure.[7] Reduced graphene oxide (rGO) with a diameter of 11 ± 4 nm could enter the nucleus of human mesenchymal stem cells (hMSCs) and cause DNA fragmentations and chromosomal aberrations even at low concentrations of 0.1 or 1.0 µg mL−1. In contrast, rGO sheets with the diameter of 3.8 ± 0.4 µm showed genotoxicity to hMSCs only at a high dose of 100 µg mL−1.[8] In general, small GO was prone to uptake by cells to produce intracellular reactive oxygen spe-cies (ROS), whereas large GO could lead to membrane stress by adsorption or insertion in addition to intracellular oxida-tive stress.[9] Considering the similar 2D structure of BP to graphene, we hypothesize that size effect will play an impor-tant role on BP’s cytotoxicity via affecting ROS generation and cell membrane disruption.

On the other hand, all the current studies of BP’s cyto-toxicity used viability reagents, e.g., methylthiazolyldiphe-nyltetrazolium bromide (MTT),[3b,c,4,5,10] cell counting kit (CCK-8),[5b,10a] and water-soluble tetrazolium (WST-8),[6] which might neglect the interactions between BP and the viability reagents. The interactions have already been dis-covered on carbon-based nanomaterials,[11] mesoporous SiO2 nanoparticles,

[12] as well as CdTe quantum dots.[13] Such interactions are very complex and tricky and could lead to either positive or negative results.[14] Recently, BP was found to reduce WST-8 and MTT assays’ reagents respectively to the orange water soluble and purple insoluble formazan products, both of which are produced by metabolically active cells, leading to the false negative results.[6] In addition, BP can also bind with the formed formazan crystals to decrease their solubility, which leads to underestimating the toxicity. Therefore, a proper toxicity assessment technique is needed to carefully evaluate BP’s cytotoxicity.

Herein, three batches of layered BP with different lat-eral size and thickness were prepared by directly exfoliating

bulk BP single crystals in oxygen-free Millipore water via the combination of ultrasonication and fractional centrifuga-tion. Their cytotoxicity toward mouse fibroblast cells (NIH 3T3), human colonic epithelial cells (HCoEpiC), and human embryonic kidney cells (293T) was determined by a label-free real-time cell analysis (RTCA) technique, which does not need any fluorescent or colorimetric viability reagents. In addition, the size effect on the possible cytotoxic mechanisms, including ROS generation and cell membrane disruption, were evaluated by the inverted fluorescence microscopy, flow cytometry, and quartz crystal microbalance with dissipation (QCM-D). This study will provide useful data for the risk evaluation and safe biomedical applications of BP.

2. Results and Discussion

2.1. Synthesis of Bulk BP Single crystals

Figure 1a showed the photo of as-grown bulk BP single crystals. The detailed synthesis process can be found in the Experimental Section. Four diffraction peaks from the (020), (040), (060), and (080) planes were observed in its X-ray diffraction (XRD) pattern (Figure 1b), indicating they are highly crystalline. The micro-Raman spectrum of the as-grown bulk BP single crystals provided insight into all sp3-bonded P allotropes (Figure 1c). It presented three characteristic peaks at 362.3, 438.6, and 466.8 cm−1, which are assigned similar to the vibrations of the A1g, B2g, and A2g phonon mode. We then conducted transmission electron microscopy (TEM) and selected-area electron diffraction (SAED) studies to evaluate the lattice structure of the BP single crystals. Figure 1d showed a typical TEM image of a phosphorene flake on copper–carbon support grid. From the high-resolution TEM image (Figure 1e), the d-spacing of this crystal plane is about 0.22 nm and perfectly matches the d002. The corresponding SAED image clearly demonstrated the single-crystalline nature of the crystals. The thermal stability of the BP single crystals was determined via thermogravim-etry and differential thermogravimetry (TG-DTG) under different ambient atmospheres (nitrogen and air flow) from room temperature to 900 °C (Figure 1f,g). Comparing these two samples, we observed that the BP single crystals appear to be more stable under the inert gases (i.e., nitrogen), fur-thermore, no sign of thermal decomposition was observed until 210 °C in the air.

2.2. Exfoliation of Layered BP

Layered BP have been obtained by liquid exfoliation in various solvents, including dimethyl sulfoxide,[15]N, N-dimethylformamide,[15,16] N-cyclohexyl-2-pyrrolidone,[17] N-methyl-2-pyrrolidone,[15] acetone,[18] isopropanol,[19] and ionic liquids.[20] Although these organic solvents can effi-ciently exfoliate the bulk BP, they are too difficult to be thor-oughly removed due to their high boiling points and strong adsorption on BP surface, thereby will affect the accuracy of cytotoxicity evaluation of layered BP. In this study, layered


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BP with diverse lateral size and thickness were exfoliated directly in oxygen-free Milli-pore water by a facile, environ-mental friendly, and scalable technique combining ultrasoni-cation and fractional centrifugation. After ultrasonication, the BP dispersions were centrifuged at different speeds to produce three batches of different size layered BP, named as BP-1, BP-2, and BP-3. The color of layered BP dispersions proceeded from dark brown to light yellow from the largest BP-1 to the smallest BP-3 (Figure 2o).

2.3. Characterizations of the As-Exfoliated Layered BP

The morphologies of layered BP were characterized by TEM (Figure 2a–c) and AFM (Figure 2d–f). The layered BP is (semi-)transparent under the electron beam, indicating their 2D structure. Statistical AFM analysis gave the average thick-ness (Figure 2g–i) of 91.9 ± 32.0, 27.0 ± 12.0, and 17.4 ± 9.1 nm, and the lateral size (Figure 2j–l) of 884.0 ± 102.2, 425.5 ± 78.8, and 208.5 ± 46.9 nm for BP-1, BP-2, and BP-3, respectively. Zeta potentials were −30.70 ± 0.53, −26.57 ± 0.35, and −11.87 ± 0.76 mV, and the average hydrodynamic size in Milli-pore water were 566.5 ± 74.7, 448.3 ± 18.6, and 175.4 ± 0.5 nm for BP-1, BP-2, and BP-3 (Figure 2m), which was increased to 1124.0 ± 107.9, 722.3 ± 67.7, and 273.3 ± 5.3 nm, respec-tively, in the 10% FBS supplemented cell culture medium (Figure 2n), probably attributed to the protein adsorption and aggregation of layered BP.

The optical properties of layered BP were analyzed by recording the UV–vis absorbance spectra under the

concentration of 40 µg mL−1. Larger/thicker BP had a broader absorbance pattern from 200 to 800 nm, whereas smaller/thinner ones absorbed strongly at shorter wave-lengths (Figure 3a). The broad absorbance was ascribed to the polydistribution of larger/thicker BP, which contributed different components to the UV spectrum. In contrast to thicker BP, more energy is needed for the electron transition in thinner BP due to their higher bandgap, thus requiring the stronger absorption in the UV region.

X-ray photoelectron spectroscopy (XPS) was used to analyze the chemical composition of layered BP. XPS survey showed two peaks at 127.7 and 126.9 eV, respec-tively assigning to 2p1/2 and 2p3/2 binding energy (Figure 3b), which are characteristics of crystalline BP.[15,16] A small peak at 130.7 eV belonged to the oxidized phosphorus (POx),

[21] which might be formed in the process of measurement.

Raman spectroscopy was used to characterize the lay-ered BP (Figure 3c). Three prominent peaks can be ascribed to one out-of-plane phonon modes (A1g) and two in-plane modes (B2g and A

2g). Also, blueshift was observed with

decreasing the size of layered BP. Compared to BP-1, A1g, and B2g peaks were blueshifted by 1.9 and 2.9 cm

−1 for BP-2, and the blueshift for BP-3 was increased to 14.5 and 16.4 cm−1. It has been reported that BP quantum dots (QDs),[22] boron nitride QDs,[23] and MoS2 QDs

[24] also showed blueshift rela-tive to their bulk forms, which was induced by the weakened interlayer van der Waals force with decreasing layer num-bers, thus releasing the atom vibration.[25] It has been known that the intensity ratio of the A1g/A

2g phonon can sensitively

reflect the degradation and oxidation state of BP. The A1g/A2

g


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Figure 1. Characterization of bulk BP single crystals. a) Photograph, b) XRD pattern, and c) micro-Raman spectrum of the BP single crystals; d) a typical TEM image of a phosphorene flake on copper–carbon support; e) High-resolution TEM image taken from the selected area (outlined in red dotted line in (d)), the inset shows the corresponding SAED pattern; the TG-DTG curves of BP powders under nitrogen flow f) and air flow g).



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Figure 2. Size and morphological characterizations of layered BP. TEM images of BP-1 a), BP-2 b), and BP-3 c); AFM images of BP-1 d), BP-2 e), and BP-3 f); statistical analysis of the thickness and lateral size of 100 pieces of BP-1 g,j), BP-2 h,k), and BP-3 i,l) measured by AFM; size distributions of layered BP in Millipore water m) and 10% FBS supplemented cell culture medium n) by DLS measurements; o) photographs of the as-exfoliated layered BP dispersions: BP-1 (left), BP-2 (middle), and BP-3 (right).

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intensity ratio of BP-1, BP-2, and BP-3 was 1.104, 1.103, and 1.013, respectively, all of which were above 0.6, confirming the basal planes of layered BP to be unoxidized.[26] Further-more, the ratio was in the sequence of BP-1 > BP-2 > BP-3, demonstrating a relatively more pronounced degradation process occurred for smaller BP, consistent with the previ-ously reported results.[17]

2.4. In Vitro Toxicity

RTCA was performed to evaluate the cytotoxicity of the as-exfoliated layered BP against three different cell lines (NIH 3T3, HCoEpiC, and 293T). The optimal cell number for each cell line (NIH 3T3: 10 000 cells per well, HCoEpiC: 3000 cells per well, and 293T: 3000 cells per well) was determined by monitoring the cell growth curve of different concentrations of cells via RTCA. A dose–response fashion of the normal-ized cell index (NCI) was evident when the concentrations of layered BP increased from 6.25 to 200 µg mL−1 (Figure S1, Supporting Information). Each layered BP exhibited various half maximal inhibitory concentrations (IC50) toward NIH 3T3, HCoEpiC, and 293T cells (Table S1, Supporting Infor-mation). The IC50 values were 3.93, 47.31, and 1.52 µg mL−1 for BP-1, and 8.15, 175.13, and 7.10 µg mL−1 for BP-2 toward the three cell lines after 24 h treatment. The smallest BP-3 had the lowest toxicity, with the IC50 values of 44.51 and 63.72 µg mL−1 to NIH 3T3 and 293T cells, respectively. Addi-tionally, BP-3 only showed moderate toxicity even at the highest dose toward HCoEpiC cells, so that the IC50 could not be determined. The IC50 of the most tolerant HCoEpiC cells was more than 30 times higher compared to that of the most sensitive 293T cells for BP-1. The cell type specific toxicity of layered BP was also observed in graphene and its deriva-tives. For example, GO dots did not pose significant toxicity to human breast cancer cells (at a dose of 50 µg mL−1)[27] or human neutral stem cells (at a dose of 250 µg mL−1),[28] but induced 50% cell death of HepG2 cells at the concentration of 12 µg mL−1.[29] Besides, Tang and co-workers found that the cytotoxicity of multiwall carbon nanotube, TiO2, and SiO2 nanoparticles also exhibited cell type specificity, with the least toxicity on NIH 3T3 cells and most toxicity on RAW macro-phages among the three tested cell lines.[30] More importantly, the IC50 followed the sequence of BP-1 < BP-2 < BP-3 for all the three cell lines (Table S1, Supporting Information) with

maximum 40 times difference between BP-1 and BP-3 for 293T cells, demonstrating the size-dependent cytotoxicity of layered BP.

The normalized cell response upon the treatment with dif-ferent concentrations of layered BP after 12, 24, and 48 h was shown in Figure 4. At each time point, the NCI of control was set at 100% and all the other NCI values were compared to the control. BP-1 exhibited severe toxic effects on 293T cells, of which only 25% was viable after 12 h exposure and 100% cell death was achieved after 24 h even at the lowest dose of 6.25 µg mL−1. In contrast, 50 µg mL−1 of BP-3 decreased the viability of 293T cells only by 15%, 25%, and 45% after 12, 24, and 48 h, respectively. As for the most tolerant HCoEpiC cells, full cell death was observed after 24 h exposure to the highest dose (200 µg mL−1) of BP-1. Moreover, 80% and 40% of HCoEpiC cells treated with 200 µg mL−1 of BP-3 remained alive at 24 and 48 h, respectively.

Interestingly, when the exposure concentration was below 1 µg mL−1, both BP-2 and BP-3 promoted the cell prolifera-tion to some degree (Figure 5 and Figure S2, Supporting Information). This was probably ascribed to the rapid deg-radation of smaller BP with the production of phosphate, which was reported to enhance the cell proliferation at high extracellular concentration.[31] Moreover, there was no evi-dent cytotoxicity induced by the degraded BP (exposing BP-3 to the air for 1 month, Figure S3, Supporting Informa-tion). It was reported that BP could be oxidized to POx and further degraded into H3PO4.

[32] According to the RTCA results (Figure S4, Supporting Information), H3PO4, under the equivalent concentration to layered BP, did not obviously inhibit the cell proliferation over the entire period of anal-ysis. Therefore, we concluded that the cytotoxicity of layered BP was attributed to its nanosize and layered structure.

2.5. AO/EB staining

The morphologies of cell apoptosis induced by layered BP were visualized by AO/EB double staining (Figure 6). HCoEpiC cells without any treatment had a uniform bright green nucleus with intact structure, indicating live cells (Figure 6a). After exposure to BP-1, some green patches or fragments, which were perinuclear chromatin condensation, appeared surrounding the green nucleus, suggesting early apoptosis (Figure 6b,c). Moreover, orange to red nucleus


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Figure 3. Characterization of layered BP. a) UV–vis absorbance of different size layered BP in Millipore water; b) XPS spectra of BP-1 after lyophilization; c) Raman spectra of the as-exfoliated layered BP in Millipore water.



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Figure 5. Percentage of cell responses of different cell lines: NIH 3T3 a), HCoEpiC b), and 293T cells c), after 24 h exposure to the layered BP (0.01–10 µg mL−1). Response data are represented by comparing to the NCI values of the respective control group.

Figure 4. Percentage of cell responses of different cell lines: NIH 3T3 a–c), HCoEpiC d–f), and 293T cells g–i), upon exposure to different sizes of layered BPs after 12 h a,d,g), 24 h b,e,h), and 48 h c,f,i) exposure to the layered BP (from 6.25 to 200 µg mL−1). Response data are represented by comparing to the NCI values of the respective control group.

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with condensed or fragmented chromatin represented late apoptosis, whereas necrotic cells were orange to red with organized structure. When treated with BP-3 (Figure 6d,e), no apoptosis was observed due to its low toxicity toward HCoEpiC cells, which is in agreement with the RTCA results above.

2.6. Intracellular ROS Generation

The intracellular ROS production was monitored by a ROS sensitive fluorophore dihydrodichlorofluorescein diacetate (DCF-DA) using inverted fluorescence microscopy and flow cytometry. An obviously elevated green fluorescence was observed in NIH 3T3 cells after exposure to layered BP com-pared to the control (Figure 7a–d), which was also quantita-

tively recorded by flow cytometry (Figure 7e,f). Compared to the control group, more than ten-fold increase of fluorescent intensity was detected in the cells treated with layered BP. In order to correlate the ROS production with the intracellular behavior of layered BP, flow cytometric light scatter analysis was performed,[33] in which the side-scattered light (SSC) was used as an indicator of the cellular uptake of nanomaterials. The intensity of SSC was significantly higher in the groups treated with layered BP than the control group (Figure S5, Supporting Information), demonstrating the intracellular density was increased due to the internalization of layered BP, which was also proved for graphene.[34] It is noteworthy that the intracellular ROS generation was not correlated to the lateral size of BP as indicated by the comparable fluo-rescent intensity of each size (Figure 7e,f), suggesting the size-dependent cytotoxicity might not be ascribed to the


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Figure 6. AO/EB staining of HCoEpiC cells after 24 h exposure of layered BP observed by inverted fluorescence microscopy. Control a), 10 µg mL−1 of BP-1 b), 50 µg mL−1 of BP-1 c), 10 µg mL−1 of BP-3 d), and 50 µg mL−1 of BP-3 e). L: live cells; EA: early apoptotic cells; LA: late apoptotic cells; N: necrotic cells.

Figure 7. Intracellular ROS detection via DCF-DA staining. NIH 3T3 cells were exposed to 10 µg mL−1 of layered BP for 4 h and visualized by inverted fluorescence microscopy: a) control, b) BP-1, c) BP-2, and d) BP-3; e,f) The fluorescence intensity were analyzed by flow cytometry.


intracellular ROS generated by layered BP. Furthermore, pre-treatment with a ROS scavenger, N-acetyl-cysteine (NAC), could partially restore the cell viability (Figure S6, Supporting Information). The viability of NIH 3T3 cells was increased to 29.8%, 42.3%, and 62.9%, respectively for BP-1, BP-2, and BP-3 due to the pretreatment of 5 × 10−3 m NAC for 1 h. These results indicated that BP-induced cytotoxicity was associated with ROS generation and there could be other mechanisms, i.e., physical damage of cell membrane, which accounted for BP’s cytotoxicity especially for the largest BP-1.

2.7. Cell Membrane Integrity

Layered structure could directly disrupt and damage cell membranes and consequently result in the release of intra-cellular contents and cell death.[35] After 4 h treatment with 10 µg mL−1 of layered BP (Figure S7, Supporting Informa-tion), BP aggregates were observed attached on the cell surface, consistent with the DLS results that layered BP aggregated in the cell culture medium. In order to detect whether the deposition of layered BP can disrupt the lipid membrane, we monitored the interactions of layered BP with the model cell membrane composed of zwitterionic

1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles in the cell culture medium by QCM-D. DOPC vesicles pre-pared in HEPES buffer was introduced into the flow cell and attained a full coverage on the QCM-D sensor, as proved by the plateauing of frequency and dissipation (Figure 8). When the cell culture medium was introduced, a decrease of frequency and increase of dissipation appeared due to the adsorption of proteins and salts on the model mem-brane. Afterward, layered BP suspended in the cell culture medium went through the system, which first raised and then decreased the frequency. In Figure 8a, BP-1 induced a sustained increase of frequency and decrease of dissipation for 20 min, which was probably ascribed to the release of water from the vesicles induced by the physical damage of the lipid membrane. In contrast, BP-2 and BP-3 raised the frequency slightly within a few minutes and then brought it down to the level lower than its starting point (Figure 8b,c), which was resulted from the deposition of layered BP on the lipid membrane. Moreover, the frequency increased by BP-3 (Figure 8c) was higher than BP-2 (Figure 8b), which could be explained by the less release of embedded water owing to the minor damage of model membrane by BP-3.

It has been reported that the cytotoxicity of GO nanosheets (a few hundred nanometer to a few micrometer)


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Figure 8. Frequency (blue) and dissipation (red) variations of the DOPC vesicles after addition of 50 µg mL−1 of BP-1 a), BP-2 b), and BP-3 c) suspended in the cell culture medium monitored by QCM-D.

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was resulted from the physical damage of cell membrane due to the direct interactions between the cell membrane and GO nanosheets.[36] In contrast, the disruption of model cell mem-brane induced by the deposition of GO (100–150 nm) could recover because of its self-healing ability.[37] In addition, Biris and co-workers demonstrated that the aggregation/agglom-eration of graphene nanosheets (100–110 nm) on the cell membrane only partially contributed to their cytotoxicity.[38] Therefore, the severe physical damage of cell membrane by larger BP could be a reasonable explanation for their higher cytotoxicity compared to the smaller ones.

3. Conclusion

The current research expanded our understanding of layered BP’s cytotoxicity that displayed a size-dependent fashion. While BP could both generate intracellular ROS and dis-rupt cell membrane integrity, only the latter was related to size effect. The larger the BP is, less membrane integrity will be retained. Since the difference in IC50 values of BP could be up to dozens of times due to the size effect and cell type specificity, we call here to pay special attention to the size of BP and the target cell lines before its application in the biomedical field. Further study is undoubtedly necessary to explore the cytotoxicity mechanisms in depth, whether other BP properties are related to cytotoxicity, and how they will affect the toxicity. Given the results from our present study, the mechanisms of BP’s cytotoxicity are strikingly compli-cated and have significant implications for the risk evaluation and safe biomedical applications of BP.

4. Experimental Section

Exfoliation Procedure of Layered BP: This study combined grinding and sonication processes to exfoliate bulk BP single crys-tals. Bulk BP (10 mg) was grounded into small pieces for 20 min using an agate mortar with a pestle. Then the BP powders were transferred to 10 mL oxygen-free Millipore water in a 25 mL sealed anaerobic bottle and sonicated with a ultrasonic cell disruption system (JY 88-11N) for 8 h at the power of 37.5 W. The ultrasound probe worked 2 s with the interval of 2 s. The temperature of sample solution was kept below 277 k by ice bath. The as-exfoliated BP suspension was centrifuged at 88 g for 20 min to remove the unex-foliated BP crystals. Then the resulting supernatant was centrifuged at 600 g for 20 min to obtain the precipitate determined as BP-1, and the supernatant was decanted gently and further centrifuged at 1800 g for 20 min to obtain BP-2. Finally, the supernatant con-tained BP-3 was separated from the precipitate (BP-2).

Cytotoxicity of Layered BP Determined by RTCA: The commonly used mammalian cell line, NIH 3T3, and two human cell lines, HCoEpiC, and 293T, were chosen to conduct the cytotoxicity exper-iments by RTCA. The untreated cells attach to the bottom of the well and increased the CI value due to the increased impedance. 50 µL of culture medium were added to each well of the E-plate 16 (ACEA Biosciences, USA) for background measurements. NIH 3T3, HCoEpiC, and 293T cells were then added at a density of 10 000, 3000, and 3000 cells per well, respectively, and incubated for

24 h. Afterward, culture medium was removed and replenished with the suspensions of different size layered BP diluted in the cell culture medium to the predetermined concentrations (6.25, 12.5, 25, 50, 100, 200 µg mL−1). Untreated cells were set as con-trol. The E-plate was incubated and monitored on the RTCA DP sys-tems (ACEA) for the continuous measurements at a time interval of 15 min. Triplicate were measured for each sample and the mean values were reported. Data analysis was performed by the RTCA software 2.0. For statistical analysis, the CI values were normal-ized to the time point of adding BP samples to vanish the cell growth difference and expressed as NCI. The IC50 was obtained by the software of the RTCA DP systems. The percentage of response was calculated by normalizing the NCI of testing group to the NCI of control group, representing as response (%) = NCI (BP sample)/NCI (control) × 100%.

AO/EB Double Staining: To visualize the cell apoptosis/necrosis, AO/EB staining kit was used to stain the cells. HCoEpiC cells were seeded on a 24 well plate at a density of 10 000 cells per well in 1 mL culture medium for 24 h incubation, then the medium was replaced with the layered BP-containing fresh culture medium (10 or 50 µg mL−1). 24 h later, the cells were washed twice with PBS, digested with trypsin, and stained with AO/EB staining kit. 10 µL of cell suspensions was dropped on the glass slide and visu-alized by an inverted fluorescence microscopy (Leica, DMI3000B, Germany).

Intracellular ROS Detection: The intracellular ROS was meas-ured by the fluorescent probe DCF-DA. 1 × 105 NIH 3T3 cells were seeded on a 6 well plate and cultured for 24 h. After 4 h treatment with 10 µg mL−1 of layered BP, the cells were rinsed with PBS for 3 times and stained with 20 × 10−6 m of DCF-DA for 30 min at 37 °C. Then the cells were imaged by an inverted fluorescence micros-copy and the fluorescence intensity was detected by NovoCyte Flow Cytometer (ACEA Biosciences, USA).

Cell Membrane Integrity: The interactions of layered BP with the model cell membrane, which was fabricated by DOPC vesi-cles, were monitored by QCM-D (E1, Q-Sense, Biolin Scientific, Sweden).[37] Before the QCM-D experiments, the gold-coated crystal sensors were immersed in a solution mixture containing H2O, H2O2 (30%), and NH3 (25%) with the ratio of 5:1:1 at 75 °C for 5 min, and then rinsed with Millipore water, followed by dry-ness under nitrogen gas stream. After cleaning, the gold-coated crystal sensors were oxidized by exposing to UV ozone for 20 min, and then rinsed with Millipore water and dried under nitrogen gas stream. The oxidation procedure was done twice.

For the preparation of DOPC vesicles, 0.2 mL (25 g L−1) of DOPC chloroform solution was dried in an Erlenmeyer flask with the ultrapure nitrogen gas stream to form a DOPC thin film at the bottom of the flask.[39] The DOPC thin film was subjected to vacuum desiccation for at least 4 h to remove the remaining chlo-roform in the DOPC layer. Then the DOPC film was resuspended by adding 5 mL HEPES buffer (10 × 10−3 m HEPES, 150 × 10−3 m NaCl, pH adjusted to 7.4) into the flask under magnetic stirring for about 30 min. The DOPC solution was extruded through a 50 nm polycarbonate membrane (Whatman) back and forth for at least 15 times using a mini-extruder (Avanti Polar Lipids Inc.) to produce unilamellar vesicles with a hydrodynamic diameter of 87–89 nm. The resulting vesicle suspension was stored in a glass vial that was sealed with nitrogen gas at 4 °C and used within 4 d after preparation.[37]



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For the QCM-D measurements, the temperature inside the QCM-D chamber was maintained at 25 °C and the liquid flow rate was kept at 100 µL min−1 for all the experiments. The shifts in frequency (the deposited mass of the deposited layer) and energy dissipation (the viscoelastic properties of the deposited layer) at the 3rd overtone were monitored simultaneously.[37] A supported vesicular layer was first formed on an oxidized gold-coated crystal sensor. Specifically, HEPES buffer was injected to pass the oxidized gold-coated crystal surface until a stable base-line was achieved (frequency signal drifts of less than 0.2 Hz per 10 min).[40] Then, 0.07 g L−1 of DOPC vesicles in HEPES buffer was injected and adhered on the crystal surface. Next, HEPES buffer was used again to remove the unadsorbed DOPC vesicles. After introducing DMEM/F12 culture media to obtain a stable baseline, 50 mg L−1 of each layered BP in DMEM/F12 was proceeded to ini-tiate the interaction process, followed by rinsing with the culture media to remove the unadsorbed BP. At the end of each experi-ment, 32 × 10−3 m Triton X-100 (a membrane solubilizer) was intro-duced to induce the rupture of the deposited vesicles.[40] All the experiments were repeated three times.

Statistical Analysis: All data are presented as the mean ± standard deviation and analyzed by the Student’s t-test. Signifi-cance level used: p < 0.05.

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements

X.Z. and Z.Z. contributed equally to this work. This work was sup-ported by the National Natural Science Foundation of China (No. 41603120), the National Key Research and Development Program of China (No. 2016YFD0800300), USDA-NIFA Hatch Pro-gram (No. MAS 00475), the National Science Foundation of China (No. 21671115), Youth Innovation Promotion Association CAS awarded to S.-Y.Z. (2017–2020), and Hundreds Talents Program of Chinese Academy of Sciences awarded to X.-J.Z. (2015–2020) and Q.Z. (2014–2019). The authors thank J. Qin in Shanghai Institutes for Biological Sciences and X. Zhang in the Institute of Metal Research for their generous supply with all the cell lines used in the project.

Conflict of Interest

The authors declare no conflict of interest.

[1] a) K. Kalantar-zadeh, J. Z. Ou, T. Daeneke, M. S. Strano, M. Pumera, S. L. Gras, Adv. Funct. Mater. 2015, 25, 5086; b) F. Bonaccorso, Z. Sun, T. Hasan, A. C. Ferrari, Nat. Photonics 2010, 4, 611; c) C. R. Dean, A. F. Young, I. Meric, C. Lee, L. Wang,

S. Sorgenfrei, K. Watanabe, T. Taniguchi, P. Kim, K. L. Shepard, J. Hone, Nat. Nano 2010, 5, 722.

[2] a) L. Li, Y. Yu, G. J. Ye, Q. Ge, X. Ou, H. Wu, D. Feng, X. H. Chen, Y. Zhang, Nat. Nanotechnol. 2014, 9, 372; b) J. Qiao, X. Kong, Z.-X. Hu, F. Yang, W. Ji, Nat. Commun. 2014, 5, 4475.

[3] a) J. Shao, H. Xie, H. Huang, Z. Li, Z. Sun, Y. Xu, Q. Xiao, X.-F. Yu, Y. Zhao, H. Zhang, H. Wang, P. K. Chu, Nat. Commun. 2016, 7, 12967; b) Z. Sun, H. Xie, S. Tang, X.-F. Yu, Z. Guo, J. Shao, H. Zhang, H. Huang, H. Wang, P. K. Chu, Angew. Chem. Int. Ed. 2015, 127, 11688; c) H. Wang, X. Yang, W. Shao, S. Chen, J. Xie, X. Zhang, J. Wang, Y. Xie, J. Am. Chem. Soc. 2015, 137, 11376.

[4] W. Chen, J. Ouyang, H. Liu, M. Chen, K. Zeng, J. Sheng, Z. Liu, Y. Han, L. Wang, J. Li, L. Deng, Y. N. Liu, S. Guo, Adv. Mater. 2017, 29, 1603864.

[5] a) W. Tao, X. Zhu, X. Yu, X. Zeng, Q. Xiao, X. Zhang, X. Ji, X. Wang, J. Shi, H. Zhang, L. Mei, Adv. Mater. 2017, 29, 1603276; b) R. Lv, D. Yang, P. Yang, J. Xu, F. He, S. Gai, C. Li, Y. Dai, G. Yang, J. Lin, Chem. Mater. 2016, 28, 4724.

[6] N. M. Latiff, W. Z. Teo, Z. Sofer, A. C. Fisher, M. Pumera, Chem. – Eur. J. 2015, 21, 13991.

[7] Y. Chang, S.-T. Yang, J.-H. Liu, E. Dong, Y. Wang, A. Cao, Y. Liu, H. Wang, Toxicol. Lett. 2011, 200, 201.

[8] O. Akhavan, E. Ghaderi, A. Akhavan, Biomaterials 2012, 33, 8017.[9] J. Ma, R. Liu, X. Wang, Q. Liu, Y. Chen, R. P. Valle, Y. Y. Zuo, T. Xia,

S. Liu, ACS Nano 2015, 9, 10498.[10] a) Y. Zhao, H. Wang, H. Huang, Q. Xiao, Y. Xu, Z. Guo, H. Xie,

J. Shao, Z. Sun, W. Han, X.-F. Yu, P. Li, P. K. Chu, Angew. Chem. Int. Ed. 2016, 128, 5087; b) C. Sun, L. Wen, J. Zeng, Y. Wang, Q. Sun, L. Deng, C. Zhao, Z. Li, Biomaterials 2016, 91, 81.

[11] J. M. Wörle-Knirsch, K. Pulskamp, H. F. Krug, Nano Lett. 2006, 6, 1261.

[12] M. Fisichella, H. Dabboue, S. Bhattacharyya, M.-L. Saboungi, J.-P. Salvetat, T. Hevor, M. Guerin, Toxicol. In Vitro 2009, 23, 697.

[13] C. Wu, L. Shi, Q. Li, H. Jiang, M. Selke, L. Ba, X. Wang, Chem. Res. Toxicol. 2010, 23, 82.

[14] F. Schrurs, D. Lison, Nat. Nanotechnol. 2012, 7, 546.[15] J. Kang, J. D. Wood, S. A. Wells, J.-H. Lee, X. Liu, K.-S. Chen,

M. C. Hersam, ACS Nano 2015, 9, 3596.[16] P. Yasaei, B. Kumar, T. Foroozan, C. Wang, M. Asadi, D. Tuschel,

J. E. Indacochea, R. F. Klie, A. Salehi-Khojin, Adv. Mater. 2015, 27, 1887.

[17] D. Hanlon, C. Backes, E. Doherty, C. S. Cucinotta, N. C. Berner, C. Boland, K. Lee, A. Harvey, P. Lynch, Z. Gholamvand, S. Zhang, K. Wang, G. Moynihan, A. Pokle, Q. M. Ramasse, N. McEvoy, W. J. Blau, J. Wang, G. Abellan, F. Hauke, A. Hirsch, S. Sanvito, D. D. O’Regan, G. S. Duesberg, V. Nicolosi, J. N. Coleman, Nat. Commun. 2015, 6, 8563.

[18] C. Hao, B. Yang, F. Wen, J. Xiang, L. Li, W. Wang, Z. Zeng, B. Xu, Z. Zhao, Z. Liu, Y. Tian, Adv. Mater. 2016, 28, 3194.

[19] C. Hao, F. Wen, J. Xiang, S. Yuan, B. Yang, L. Li, W. Wang, Z. Zeng, L. Wang, Z. Liu, Y. Tian, Adv. Funct. Mater. 2016, 26, 2016.

[20] W. Zhao, Z. Xue, J. Wang, J. Jiang, X. Zhao, T. Mu, ACS Appl. Mater. Interfaces 2015, 7, 27608.

[21] J. D. Wood, S. A. Wells, D. Jariwala, K.-S. Chen, E. Cho, V. K. Sangwan, X. Liu, L. J. Lauhon, T. J. Marks, M. C. Hersam, Nano Lett. 2014, 14, 6964.

[22] J. T. Robinson, S. M. Tabakman, Y. Liang, H. Wang, H. S. Casalongue, D. Vinh, J. Am. Chem. Soc. 2011, 133, 6825.

[23] L. Lin, Y. Xu, S. Zhang, I. M. Ross, A. C. M. Ong, D. A. Allwood, Small 2014, 10, 60.

[24] V. Stengl, J. Henych, Nanoscale 2013, 5, 3387.[25] H. Li, Q. Zhang, C. C. R. Yap, B. K. Tay, T. H. T. Edwin, A. Olivier,

D. Baillargeat, Adv. Funct. Mater. 2012, 22, 1385.[26] A. Favron, E. Gaufres, F. Fossard, A.-L. Phaneuf-Lheureux,

N. Y. W. Tang, P. L. Levesque, A. Loiseau, R. Leonelli, S. Francoeur, R. Martel, Nat. Mater. 2015, 14, 826.

full papers



small 2017, 13, 1701210

[27] J. Peng, W. Gao, B. K. Gupta, Z. Liu, R. Romero-Aburto, L. Ge, L. Song, L. B. Alemany, X. Zhan, G. Gao, S. A. Vithayathil, B. A. Kaipparettu, A. A. Marti, T. Hayashi, J.-J. Zhu, P. M. Ajayan, Nano Lett. 2012, 12, 844.

[28] W. Shang, X. Zhang, M. Zhang, Z. Fan, Y. Sun, M. Han, L. Fan, Nanoscale 2014, 6, 5799.

[29] R. Gokhale, P. Singh, Part. Part. Syst. Charact. 2014, 31, 433.

[30] S. K. Sohaebuddin, P. T. Thevenot, D. Baker, J. W. Eaton, L. Tang, Part. Fibre Toxicol. 2010, 7, 22.

[31] M. C. Roussanne, M. Lieberherr, J. C. Souberbielle, E. Sarfati, T. Drüeke, A. Bourdeau, Eur. J. Clin. Invest. 2001, 31, 610.

[32] M. Serrano-Ruiz, M. Caporali, A. Ienco, V. Piazza, S. Heun, M. Peruzzini, Adv. Mater. Interfaces 2016, 3, 1500441.

[33] H. Suzuki, T. Toyooka, Y. Ibuki, Environ. Sci. Technol. 2007, 41, 3018.

[34] W. Hu, C. Peng, M. Lv, X. Li, Y. Zhang, N. Chen, ACS Nano 2011, 5, 3693.

[35] a) Y. Zhang, S. F. Ali, E. Dervishi, Y. Xu, Z. Li, D. Casciano, ACS Nano 2010, 4, 3181; b) G. Duan, S. G. Kang, X. Tian, J. A. Garate, L. Zhao, C. Ge, Nanoscale 2015, 7, 115214.

[36] W. Hu, C. Peng, M. Lv, X. Li, Y. Zhang, N. Chen, C. Fan, Q. Huang, ACS Nano 2011, 5, 3693.

[37] X. Liu, K. L. Chen, Langmuir 2015, 31, 12076.[38] Y. Zhang, S. F. Ali, E. Dervishi, Y. Xu, Z. Li, D. Casciano, A. S. Biris,

ACS Nano 2010, 4, 3181.[39] A. Carnini, T. T. Nguyen, D. T. Cramb, Can. J. Chem. 2007, 85, 513.[40] X. Liu, K. L. Chen, Environ. Sci.: Nano 2016, 3, 146.

Received: April 15, 2017Revised: May 28, 2017Published online: July 11, 2017

size effect on the cytotoxicity of layered black phosphorus...

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