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Detection of Endotoxin Contamination of Graphene Based Materials Using the TNF- α Expression Test and Guidelines for Endotoxin-Free Graphene Oxide Production

View Article: PubMed Central - PubMed

ABSTRACT

Nanomaterials may be contaminated with bacterial endotoxin during production and handling, which may confound toxicological testing of these materials, not least when assessing for immunotoxicity. In the present study, we evaluated the conventional Limulus amebocyte lysate (LAL) assay for endotoxin detection in graphene based material (GBM) samples, including graphene oxide (GO) and few-layered graphene (FLG). Our results showed that some GO samples interfered with various formats of the LAL assay. To overcome this problem, we developed a TNF-α expression test (TET) using primary human monocyte-derived macrophages incubated in the presence or absence of the endotoxin inhibitor, polymyxin B sulfate, and found that this assay, performed with non-cytotoxic doses of the GBM samples, enabled unequivocal detection of endotoxin with a sensitivity that is comparable to the LAL assay. FLG also triggered TNF-α production in the presence of the LPS inhibitor, pointing to an intrinsic pro-inflammatory effect. Finally, we present guidelines for the preparation of endotoxin-free GO, validated by using the TET.

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TNF-α Expression Test (TET) for endotoxin detection.a). The TET was performed to detect endotoxin content in all GBMs upon exposure of HMDM to 25 and 50 μg/ml for 24 h. The differences in TNF-α expression in the presence and absence of polymyxin B sulfate (Poly-B) (10 μM) provided evidence of the presence of endotoxin in the GO-A, GO-D and FLG samples. Such differences were not observed upon exposure of cells to GO-B and GO-C. One-way Anova with post hoc Turkey’s test was performed to analyze the statistical significance between the sample exposed with and without Poly-B. Note that also triggered significant production of TNF-α in presence of Poly-B. b) Standard curve showing relationship between LPS and TNF-α expression. Poly-B blocked LPS-triggered TNF-α production, as expected. Experiments were conducted using cells from at least three independent donors per experiment. (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001).
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pone.0166816.g004: TNF-α Expression Test (TET) for endotoxin detection.a). The TET was performed to detect endotoxin content in all GBMs upon exposure of HMDM to 25 and 50 μg/ml for 24 h. The differences in TNF-α expression in the presence and absence of polymyxin B sulfate (Poly-B) (10 μM) provided evidence of the presence of endotoxin in the GO-A, GO-D and FLG samples. Such differences were not observed upon exposure of cells to GO-B and GO-C. One-way Anova with post hoc Turkey’s test was performed to analyze the statistical significance between the sample exposed with and without Poly-B. Note that also triggered significant production of TNF-α in presence of Poly-B. b) Standard curve showing relationship between LPS and TNF-α expression. Poly-B blocked LPS-triggered TNF-α production, as expected. Experiments were conducted using cells from at least three independent donors per experiment. (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001).

Mentions: Based on the results of the Alamar Blue assay, we selected 25 and 50 μg/ml for endotoxin evaluation by TET. The TET was performed with GBMs in the presence or absence of the endotoxin inhibitor polymyxin B sulfate [25] and LPS was included as a positive control. If HMDMs exposed to GO produce TNF-α and if the levels of TNF-α are equivalent in the presence or absence of polymyxin B sulfate, then TNF-α production is an intrinsic feature of the GBM. While if HMDM express less TNF-α upon exposure to GBM in the presence of polymyxin B sulfate then the GBM is endotoxin contaminated. If there is no secretion of TNF-α, then there is no endotoxin present (Fig 1). HMDM were thus exposed for 24 h to 25 and 50 μg/ml of GBMs in the presence or absence of 10 μM polymyxin B sulfate. Then, supernatants were collected and TNF-α was measured using a specific ELISA. In addition, HMDM were exposed to different doses of LPS (100 ng/ml to 10 pg/ml) to generate a standard curve. The TET showed that GO-A triggered a moderate, albeit significant production of TNF-α in macrophages which was suppressed in the presence of polymyxin B, indicating that GO-A was, in fact, endotoxin-contaminated (Fig 4A). Based on the standard curve shown in Fig 4B, 50 μg/ml GO-A is concluded to contain 30 pg/ml LPS. The results for GO-B are discussed below. The commercial GO-C sample did not trigger TNF-α production in macrophages at 25 or 50 μg/ml (with or without polymyxin B). On the other hand, the commercial GO-D sample triggered a minor, but statistically significant production of TNF-α which was suppressed in the presence of polymyxin B, indicating that this sample contained endotoxin. Finally, FLG, triggered significant TNF-α production both in the presence and absence of polymyxin B, although in the presence of the endotoxin inhibitor, the level of TNF-α production was substantially reduced (Fig 4A). This result clearly indicates a) that FLG was endotoxin contaminated, and b) that FLG has an inherent propensity to trigger pro-inflammatory cytokine production. Thus, even in the presence of polymyxin B, some GBMs (eg., GO-A and GO-D) induced a low, but detectable level of TNF-α production while a significant level of TNF-α production was noted for FLG. Hence, the TET assay, conducted in the absence or presence of an endotoxin inhibitor to exclude endotoxin mediated effects, has revealed the intrinsic pro-inflammatory properties of certain GBMs. Qu et al. reported previously that GO induced necrotic cell death in murine macrophages and this was suggested to be mediated through autocrine TNF-α signaling in these cells [19]. The authors also argued that the effects of GO were mediated by activation of TLR4, a pattern recognition receptor that serves as a key sensor of endotoxin. However, it should be noted that endotoxin contamination of the test materials could yield ambiguous results and experiments conducted with or without polymyxin B could help to address this. Furthermore, although GO-A and GO-D induced statistically significant levels of TNF-α production in the presence of polymyxin B, the TNF-α levels remained very low (below 50 pg/50.000 cells) (Fig 4A). GO-C, on the other hand, did not induce any TNF-α secretion. Comparing the lateral dimensions of the GO samples (Table 1), it appears that large flakes (i.e., GO-A and GO-D) are capable of inducing low, but statistically significant TNF-α production, while small flakes (GO-C) did not elicit such effects. This result is in accordance with previous studies that have pointed to a crucial role of the lateral dimensions of GO in activating macrophages and stimulating pro-inflammatory effects [29]. FLG induced marked TNF-α production in the presence of polymyxin B (about 500 pg/50.000 cells) (Fig 4A) and induced dose-dependent cytotoxicity (Fig 3C) while the GO samples were non-cytotoxic, suggesting, overall, that GO is less toxic than FLG.


Detection of Endotoxin Contamination of Graphene Based Materials Using the TNF- α Expression Test and Guidelines for Endotoxin-Free Graphene Oxide Production
TNF-α Expression Test (TET) for endotoxin detection.a). The TET was performed to detect endotoxin content in all GBMs upon exposure of HMDM to 25 and 50 μg/ml for 24 h. The differences in TNF-α expression in the presence and absence of polymyxin B sulfate (Poly-B) (10 μM) provided evidence of the presence of endotoxin in the GO-A, GO-D and FLG samples. Such differences were not observed upon exposure of cells to GO-B and GO-C. One-way Anova with post hoc Turkey’s test was performed to analyze the statistical significance between the sample exposed with and without Poly-B. Note that also triggered significant production of TNF-α in presence of Poly-B. b) Standard curve showing relationship between LPS and TNF-α expression. Poly-B blocked LPS-triggered TNF-α production, as expected. Experiments were conducted using cells from at least three independent donors per experiment. (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001).
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pone.0166816.g004: TNF-α Expression Test (TET) for endotoxin detection.a). The TET was performed to detect endotoxin content in all GBMs upon exposure of HMDM to 25 and 50 μg/ml for 24 h. The differences in TNF-α expression in the presence and absence of polymyxin B sulfate (Poly-B) (10 μM) provided evidence of the presence of endotoxin in the GO-A, GO-D and FLG samples. Such differences were not observed upon exposure of cells to GO-B and GO-C. One-way Anova with post hoc Turkey’s test was performed to analyze the statistical significance between the sample exposed with and without Poly-B. Note that also triggered significant production of TNF-α in presence of Poly-B. b) Standard curve showing relationship between LPS and TNF-α expression. Poly-B blocked LPS-triggered TNF-α production, as expected. Experiments were conducted using cells from at least three independent donors per experiment. (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001).
Mentions: Based on the results of the Alamar Blue assay, we selected 25 and 50 μg/ml for endotoxin evaluation by TET. The TET was performed with GBMs in the presence or absence of the endotoxin inhibitor polymyxin B sulfate [25] and LPS was included as a positive control. If HMDMs exposed to GO produce TNF-α and if the levels of TNF-α are equivalent in the presence or absence of polymyxin B sulfate, then TNF-α production is an intrinsic feature of the GBM. While if HMDM express less TNF-α upon exposure to GBM in the presence of polymyxin B sulfate then the GBM is endotoxin contaminated. If there is no secretion of TNF-α, then there is no endotoxin present (Fig 1). HMDM were thus exposed for 24 h to 25 and 50 μg/ml of GBMs in the presence or absence of 10 μM polymyxin B sulfate. Then, supernatants were collected and TNF-α was measured using a specific ELISA. In addition, HMDM were exposed to different doses of LPS (100 ng/ml to 10 pg/ml) to generate a standard curve. The TET showed that GO-A triggered a moderate, albeit significant production of TNF-α in macrophages which was suppressed in the presence of polymyxin B, indicating that GO-A was, in fact, endotoxin-contaminated (Fig 4A). Based on the standard curve shown in Fig 4B, 50 μg/ml GO-A is concluded to contain 30 pg/ml LPS. The results for GO-B are discussed below. The commercial GO-C sample did not trigger TNF-α production in macrophages at 25 or 50 μg/ml (with or without polymyxin B). On the other hand, the commercial GO-D sample triggered a minor, but statistically significant production of TNF-α which was suppressed in the presence of polymyxin B, indicating that this sample contained endotoxin. Finally, FLG, triggered significant TNF-α production both in the presence and absence of polymyxin B, although in the presence of the endotoxin inhibitor, the level of TNF-α production was substantially reduced (Fig 4A). This result clearly indicates a) that FLG was endotoxin contaminated, and b) that FLG has an inherent propensity to trigger pro-inflammatory cytokine production. Thus, even in the presence of polymyxin B, some GBMs (eg., GO-A and GO-D) induced a low, but detectable level of TNF-α production while a significant level of TNF-α production was noted for FLG. Hence, the TET assay, conducted in the absence or presence of an endotoxin inhibitor to exclude endotoxin mediated effects, has revealed the intrinsic pro-inflammatory properties of certain GBMs. Qu et al. reported previously that GO induced necrotic cell death in murine macrophages and this was suggested to be mediated through autocrine TNF-α signaling in these cells [19]. The authors also argued that the effects of GO were mediated by activation of TLR4, a pattern recognition receptor that serves as a key sensor of endotoxin. However, it should be noted that endotoxin contamination of the test materials could yield ambiguous results and experiments conducted with or without polymyxin B could help to address this. Furthermore, although GO-A and GO-D induced statistically significant levels of TNF-α production in the presence of polymyxin B, the TNF-α levels remained very low (below 50 pg/50.000 cells) (Fig 4A). GO-C, on the other hand, did not induce any TNF-α secretion. Comparing the lateral dimensions of the GO samples (Table 1), it appears that large flakes (i.e., GO-A and GO-D) are capable of inducing low, but statistically significant TNF-α production, while small flakes (GO-C) did not elicit such effects. This result is in accordance with previous studies that have pointed to a crucial role of the lateral dimensions of GO in activating macrophages and stimulating pro-inflammatory effects [29]. FLG induced marked TNF-α production in the presence of polymyxin B (about 500 pg/50.000 cells) (Fig 4A) and induced dose-dependent cytotoxicity (Fig 3C) while the GO samples were non-cytotoxic, suggesting, overall, that GO is less toxic than FLG.

View Article: PubMed Central - PubMed

ABSTRACT

Nanomaterials may be contaminated with bacterial endotoxin during production and handling, which may confound toxicological testing of these materials, not least when assessing for immunotoxicity. In the present study, we evaluated the conventional Limulus amebocyte lysate (LAL) assay for endotoxin detection in graphene based material (GBM) samples, including graphene oxide (GO) and few-layered graphene (FLG). Our results showed that some GO samples interfered with various formats of the LAL assay. To overcome this problem, we developed a TNF-α expression test (TET) using primary human monocyte-derived macrophages incubated in the presence or absence of the endotoxin inhibitor, polymyxin B sulfate, and found that this assay, performed with non-cytotoxic doses of the GBM samples, enabled unequivocal detection of endotoxin with a sensitivity that is comparable to the LAL assay. FLG also triggered TNF-α production in the presence of the LPS inhibitor, pointing to an intrinsic pro-inflammatory effect. Finally, we present guidelines for the preparation of endotoxin-free GO, validated by using the TET.

No MeSH data available.


Related in: MedlinePlus