Oxidative State and Lateral Size of Graphene Oxide Determine Differential Cell Death Mechanisms and Pro-Inflammatory Responses in the Liver
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摘要: 氧化石墨烯(GOs)在药物传递、生物传感和生物成像等方面表现出极佳的性能，但在肝脏等器官的生物相容性方面仍存在问题。分别合成了大(约510 nm)、小(约110 nm)两种尺寸的原始氧化态氧化石墨烯（pGO，根据尺寸不同分别命名为pGO-L和pGO-S）和还原态氧化石墨烯（rGO，根据尺寸不同分别命名为rGO-L和rGO-S）；探讨了GOs对3种肝细胞（枯氏细胞、肝窦内皮细胞、肝细胞）的生物学影响。结果表明，pGO能诱导枯氏细胞膜损伤并引起其坏死(pGO-L>pGO-S)，而对肝窦内皮细胞和肝细胞毒性较小；rGO可诱导枯氏细胞和肝窦内皮细胞凋亡(rGO-L>rGO-S)，但对肝细胞影响微乎其微。在溶酶体和NLRP3炎症小体水平上进一步研究GOs的胞内效应，结果表明，rGO造成枯氏细胞和肝窦内皮细胞的溶酶体损伤并诱导了白细胞介素-1β（IL-1β）的产生，但rGO在肝细胞中与pGO组实验中均未见类似效应。研究表明GOs的表面氧化状态和横向尺寸在肝脏细胞中引发了不同的细胞死亡机制和炎症反应。Abstract: Two-dimensional (2D) graphene oxide (GO) exhibits superlative properties for biomedical applications such as drug delivery, biosensing and bioimaging. There are questions, however, about their biocompatibility on organs including liver, which is frequently targeted by therapeutic nanomaterials or secondary spread from other uptake sites. In this study, we systemically explored the biological impact of GO on three major liver cell types: Kupffer cells, liver sinusoidal endothelial cells (LSEC), and hepatocytes. Pristine (pGO) and reduced (rGO) oxidation states of GO flakes with large (510 nm) and small sizes (110 nm) were synthesized. These samples show differences in cellular response, depending on oxidation state as well as lateral size. While pGO induced plasma membrane damage and necrosis (large > small flakes) in Kupffer cells, minimal cytotoxicity was observed in LSEC and hepatocytes. In contrast, rGO induced apoptosis in Kupffer cells (large > small flakes) and LSEC, with negligible effects in hepatocytes. While pGO attached to the Kupffer cell membrane and induced lipid peroxidation and cytoskeleton disruption, rGO was mostly taken up in Kupffer cells. Moreover, while pGO and rGO were taken up in roughly similar amounts by LSEC, little uptake for hepatocytes. Further study of intracellular effects of the GO species at the level of lysosomes and functionally linked NLRP3 inflammasomes demonstrated that lysosomal damage by rGO in Kupffer and LSEC induced IL-1β production. Similar effects were not seen in hepatocytes or with pGO. Collectively, this study reveals that surface oxidation state and lateral size of GO differentially impact liver cells based on interactions with cell membranes, cellular uptake, and damage to lysosomes.
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Key words:
- Kupffer cell /
- LSEC /
- hepatocytes /
- necrosis /
- apoptosis /
- NLRP3 inflammasome
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图 1 GOs材料理化性质的表征
Figure 1. Physicochemical characterizations of GOs materials
(a) Determination of the flake sizes of pGO and rGO nanosheets by AFM; (b) XPS spectra of surface functional groups on pGO and rGO nanosheets; (c) Quantification of oxygen-to-carbon mass ratio (m(O)∶m(C)) and the massfractions of oxidized groups, respectively; (d) Raman spectra with the wavelength region of 1000—2000 cm−1 and ID/IG ratio of pGO and rGO samples; (e) Contact angle of pGO and rGO nanosheets. Data represent the mean ± standard error from triplicates
图 2 GOs在 Kup5、 SK-HEP-1 和 Hepa 1-6 细胞中的毒性实验
Figure 2. Cytotoxicity of GOs in Kup5, SK-HEP-1 and Hepa 1-6 cells
(a) Assessment of cell death in three cell types by the CellToxTM Green assay; (b) Assessment of cell viability in three cell types by the ATP liteTM assay. ZnO (D=(22.6 ± 5.1) nm) nanoparticles were used as a positive control for cell death. * P<0.05 compared to cell control based on student's T Test. & stands for P<0.05 compared pGO-S with pGO-L based on student’s T Test. # stands for P<0.05 compared rGO-S with rGO-L based on student’s T Test; (c) Heat map summary of GO materials induced cytotoxicity in three cell types based on the proportion of cell death in three cell types obtained from Cell Tox assay, respectively
图 3 GOs诱导3种肝脏细胞死亡的机制
Figure 3. Mechanisms of cell death for three types of liver cells inducedby GOs
(a) three types of liver cells after treatment with pGO and rGO nanosheets for 16 h, respectively. The assay is Annexin V/PI staining evaluated by flow cytometry. (b) Caspase-3/Caspase-7 activities induced by pGO and rGO nanosheets in Kup5 and SK-HEP-1 cells, respectively. The concentration for both pGO and rGO was 50 μg/mL and the incubation time was 16 h. * P<0.05 compared to cell control based on student's T Test. (c) Differential interference contrast (DIC) images of cells (Kup5, SK-HEP-1 and Hepa 1-6) after treatment with pGO or rGO for 24 h. Materials’ concentrations are 50 μg/mL, respectively. (d) Confocal images of pGO-L induced cytoskeleton disruption in Kup5 after 12 h incubation, respectively. Cell cytoskeleton and membranes were stained with AF633 conjugated phalloidin and WGA AF488 conjugated wheat germ agglutinin, respectively. The images are shown at single Z-plane
图 4 GOs与肝脏细胞的细胞结合实验
Figure 4. Cellular association of GOs with liver cells
Cellular association of pGO and rGO nanosheets in (a) Kup5, (b) SK-HEP-1 and (c) Hepa 1-6 cell lines by fluorescence confocal microscopy and flow cytometry. Cells were treated with 50 μg/mL pGO and rGO nanosheets for 16 h, respectively. Cell nuclei and membranes were stained with Hoechst 33342 and Alexa Fluor-594 conjugated wheat germ agglutinin, respectively. The images are shown at single Z-plane.Flow cytometry side scattering (SSC) data (bottom right figures) show the percentage of cellular association of pGO and rGO (50 μg/ mL) after 16 h incubation at 37 °C. (d) TEM images of cellular interaction of pGO-L and rGO-L nanosheets in Kup5 cells after 6 h incubation. Right two images of the magnification of the frame area in the middle two image, respectively. (e) Cellular association of GOs (50 μg/mL) by SK-HEP-1 and Hepa 1-6 with (red bars) or without (blue bars) BLT-1 treatment. Cells were pre-treated with BLT-1 for 8 h prior to pGO/rGO exposure at a working concentration of 100 nmol/L. Cellular uptake were quantified by side scatter of flow cytometry
图 5 GOs诱导肝脏细胞脂质过氧化实验
Figure 5. Lipid peroxidation test of liver cells induced by GOs
Confocal imaging (up) and percentage (down) of GOs induced lipid peroxidation in (a) Kup 5, (b) SK-HEP-1 and (c) Hepa 1-6 cells, respectively. To assess lipid peroxidation, cells were exposed to 25 μg/mL pGO or rGO nanosheets (for 12 h) or 20 μmol/L cumene hydroperoxide (CH) as a positive control (for 1.5 h), and then stained with the Image-iT lipid peroxidation reagent and Hoechst 33342 (nuclei staining), respectively. The lipid peroxidation was determined by the reduction and oxidation of the Image-iT sensor at excitation/emission wavelengths of 581 nm/591 nm (Texas Red, pristine sensor) and 488 nm/510 nm (FITC, oxidized sensor). Quantification of cells with lipid peroxidation was done using FITC channel of flow cytometry, respectively. Data represent the mean ± standard error from two independent experiments, and at least 10000 cells were analyzed in each experiment. * stands for P<0.05 compared to cell control based on student's T Test
图 6 Kup5细胞在pGO和rGO（50 μg/mL）中处理6 h后溶酶体损伤及组织蛋白酶B释放
Figure 6. Lysosomal damage and cathepsin B release in Kup5 cells after pGO and rGO (50 μg/mL) treatment for 6 h, respectively
Cathepsin B was stained with Magic Red at 26 nmol/L for 2 h; Monosodium urate crystals (MSU) was used a positive control for lysosome damage
图 7 Kup5，SK-HEP-1 和 Hepa 1-6三种肝脏细胞暴露于pGO和rGO（50 μg/mL）24 h 后 IL-1β的释放
Figure 7. IL-1β production induced by pGO and rGO (50 μg/mL) in Kup5, SK-HEP-1, and Hepa 1-6 after 24 h incubation, respectively
Data represent the mean ± standard error from three independent experiments; * P<0.05 compared to cell control based on student's T test; # P<0.05 based on student ’s T test
图 8 GOs 诱导Kup5，SK-HEP-1 和 Hepa 1-6 三种肝脏细胞死亡和促炎症反应机理示意图
Figure 8. Schematic illustration of GO materials induced differential cell death mechanisms and pro-inflammatory responses in Kup5, SK-HEP-1, and Hepa 1-6 cells
表 1 pGO和rGO纳米片在细胞培养基中的水合粒经和ζ电位
Table 1. Hydrodynamic size and ζ potential of pGO and rGO nanosheets in different cell culture media
Medium Hydrodynamic size/nm Zeta potential/mV pGO-S pGO-L rGO-S rGO-L pGO-S pGO-L rGO-S rGO-L Kup5 258.9 ± 6.6 448.4 ± 19.2 383.7 ± 52.5 685.1 ± 37.0 −13.8 ± 1.6 −11.9 ± 2.6 −12.9 ± 2.5 −12.7 ± 1.0 SK-HEP-1 256.4 ± 7.2 556.8 ± 6.3 352.4 ± 7.3 557.9 ± 16.0 −14.7 ± 2.3 −14.4 ± 1.2 −14.9 ± 2.0 −14.7 ± 3.6 Hepa 1-6 278.5 ± 5.0 448.6 ± 8.1 370.0 ± 12.3 765.6 ± 20.5 −14.4 ± 2.6 −14.6 ± 4.2 −14.3 ± 2.9 −13.7 ± 1.6 
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表 2 pGO和rGO在酶体模拟液（pH 4.5）6 h后的水合粒径
Table 2. Hydrodynamic sizes of pGO and rGO after suspending in phagolysosomal simulant fluid (pH 4.5) for 6 h, respectively
GOs Hydrodynamic size/nm PDI pGO-S 829.8 ± 80.9 0.378 pGO-L 1479.8 ± 25.9 0.375 rGO-S 12215.2 ± 896.4 0.472 rGO-L 16214.4 ± 8099.8 r0.476 Original sizes of GOs are shown in brackets, respectively
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