Effect of Oxidative State and Lateral Size of Graphene Oxide on Cell Death Mechanisms and Pro-Inflammatory Responses in the Liver
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摘要: 氧化石墨烯(GO)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GO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GO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在肝细胞中和pGO实验组中均未见类似效应。研究表明GO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) has been widely used for biomedical applications such as drug delivery, biosensing and bioimaging. However, problems still exist with respect to their toxicity to major organs such as the liver. In this study, we systemically investigated the impact of GO on three major liver cell types: Kupffer cells, liver sinusoidal endothelial cells (LSEC), and hepatocytes. Pristine and reduced GO, nanosheets (pGO and rGO) with two sizes (about 510 nm and 110 nm) were synthesized. These materials elicited different cellular responses depending on the oxidation state as well as lateral size of GO. While pGO induced plasma membrane damage and necrosis (large nanosheets > small nanosheets) in Kupffer cells, minimal cytotoxicity was observed in LSEC and hepatocytes. In contrast, rGO induced apoptosis in Kupffer cells (large nanosheets > small nanosheets) and LSEC, with negligible effects in hepatocytes. While pGO could attach to the Kupffer cell membrane and induce 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 was internalized for hepatocytes. Studies on the intracellular effects of the GO species at the lysosomal and inflammasomal level demonstrated that rGO damaged lysosomes and induced IL-1β production in both Kupffer and LSEC cells, while similar effects were absent in hepatocytes or with pGO. Our research reveals that the surface oxidation state and lateral size of GO determine their biological effect on tuning the fate of liver cells.
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Key words:
- Kupffer cell /
- LSEC /
- hepatocyte /
- necrocytosis /
- apoptosis /
- NLRP3 inflammasome
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图 2 GO在 Kup5、 SK-HEP-1 和 Hepa 1-6 细胞中的毒性实验
Figure 2. Cytotoxicity of GO in Kup5, SK-HEP-1 and Hepa 1-6 cells
* Stands for 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
图 3 GO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 induced by GO
图 4 GO与肝脏细胞的细胞结合实验
Figure 4. Cellular association of GO 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. (d) TEM images of cellular interaction of pGO-L and rGO-L nanosheets in Kup5 cells after 6 h incubation. Two images of the magnification on the right are the frame area in the middle two images, respectively. (e) Cellular association of GO (50 μg/mL). 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 was quantified by side scatter of flow cytometry
图 5 GO诱导肝脏细胞脂质过氧化实验
Figure 5. Lipid peroxidation test of liver cells induced by GO
Confocal imaging (up) and percentage (down) of GO induced lipid peroxidation in (a) Kup 5, (b) SK-HEP-1 and (c) Hepa 1-6 cells, respectively. * 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 treated by pGO and rGO (50 μg/mL) for 6 h, respectively
图 7 Kup5，SK-HEP-1 和 Hepa 1-6三种肝脏细胞暴露于pGO和rGO 24 h 后 IL-1β的释放
Figure 7. IL-1β production induced by pGO and rGO in Kup5, SK-HEP-1, and Hepa 1-6 after 24 h incubation, respectively
* Stands for P<0.05 compared to cell control based on student's T test; # Stands for P<0.05 based on student ’s T test
图 8 GO 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 induced differential cell death mechanisms and pro-inflammatory responses in Kup5, SK-HEP-1, and Hepa 1-6 cells
表 1 GO材料的氧碳质量比和氧化基团质量分数
Table 1. Quantification of oxygen-to-carbon mass ratio and the mass fractions of oxidized groups of GO materials
GO m(O) : m(C) w(C−C)/% w(C−O)/% w(C=O )/% w(O−C=O )/% pGO-S 1.0 : (2.2 ± 0.1) 48.5 ± 2.2 44.7 ± 1.8 6.8 ± 1.1 − pGO-L 1.0 : (2.1 ± 0.1) 53.8 ± 1.1 39.7 ± 0.8 6.5 ± 0.4 − rGO-S 1.0 : (6.7 ± 0.1) 63.0 ± 2.0 16.7 ± 0.9 14.9 ± 1.2 5.4 ± 0.1 rGO-L 1.0 : (7.7 ± 0.1) 64.1 ± 0.7 15.5 ± 0.7 15.2 ± 0.7 5.1 ± 0.2 
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表 2 pGO和rGO纳米片在细胞培养基中的水合粒径和Zeta电位
Table 2. Hydrodynamic size and Zeta 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 3 pGO和rGO在溶酶体模拟液（pH 4.5）中6 h后的水合粒径
Table 3. Hydrodynamic sizes of pGO and rGO after suspending in lysosomal simulant fluid (pH 4.5) for 6 h, respectively
GO 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 0.476
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