Carbon Nanotubes Activate Limulus Amebocyte Lysate Coagulation

Letter www.acsami.org

Carbon Nanotubes Activate Limulus Amebocyte Lysate Coagulation by Interface Adsorption Man Yang,†,‡ Xin Nie,†,§ Jie Meng,*,§ Jian Liu,§ Zhiwei Sun,‡ and Haiyan Xu*,§ ‡

School of Public Health, Capital Medical University, Beijing 100069, P. R. China Institute of Basic Medical Sciences Chinese Academy of Medical Sciences & School of Basic Medicine, Peking Union Medical College, Beijing 100005, P. R. China

§

S Supporting Information *

ABSTRACT: Limulus amebocyte lysate (LAL) assay is worldwide requested in the assessment of endotoxin contamination for biomaterials. As carbon nanotubes are one major nanomaterial with multiple potentials in biomedical application, here we investigate whether oxidized multiwalled carbon nanotubes (OMWCNT) interferes the assessment by LAL assays. We showed that the endotoxin free O-MWCNT dispersing in aqueous solutions could activate both the gel-clotting and the end-point chromogenic LAL assay by converting coagulogen into coagulin through interfacial interactions between O-MWCNT and enzymes in the assays. In conclusion, the O-MWCNT could induce false positive results by activating the enzyme cascade of LAL. KEYWORDS: carbon nanotubes, endotoxin contamination, limulus amebocyte lysate, interface interaction, protein adsorption, enzyme activation

C

assay is easily disturbed in the presence of TiO2, Ag, CaCO3 and SiO2 nanoparticles, suggesting the assay was not suitable at high particle concentrations.18 So far mechanisms behind the observed interference events of nanomaterials to LAL assay have not been given adequate investigations. Furthermore, few studies have clarified the direct interaction of the LAL with CNTs. Hence the aim of this work was to investigate whether CNTs affected the performance of gel-clotting or end point chromogenic LAL assays and reveal what mechanisms are underlying. We showed that the endotoxin-free oxidized multiwalled carbon nanotubes that dispersed in the aqueous solution (termed as O-MWCNT-aq) could activate LAL assay, giving false positive results. In this work, O-MWCNT was prepared according to the previous report19 and treated at 250 °C for 45 min to eliminate any possible endotoxin prior to be dispersing in water. The OMWCNT-aq was stable without obvious sediment for at least 2 weeks. To observe O-MWCNT in the aqueous solution, a drop of the solution was dripped on the silicone substrate and allowed for dry, then was subjected to scanning electron microscopy (SEM). The O-MWCNT showed characteristic tubelike structures (Figure 1a) with the length ranging from 0.2 to 3 μm that was counted statistically from 10 SEM images. A characteristic absorption at 260 nm was observed and the intensity of the absorbance was dependent on the concen-

arbon nanotubes (CNTs) are one of the most important nanomaterials with a steady increase in production and applications,1 because they not only display unique mechanical, chemical, and electrical properties but can also be further modified or functionalized with varieties of chain moieties, attracting intensive investigations to explore various biomedical applications including immunological adjuvant,2−4 delivery systems of therapeutic agents,5−7 diagnostic probes8−10 and scaffolds for inducing tissue regeneration.11,12 Prior to biomedical applications, CNTs have to be examined whether they are pyrogenic substances free, especially bacterial endotoxins.13 Therefore, accurate detection and clarification of endotoxin contamination with CNTs are of crucial roles as well as significance. The LAL is the aqueous extract of wild horseshoe crabs (Limulus polyphemus or Trachypleustridentatus), which contains all the components necessary to perform a coagulation cascade. Generally, the presence of endotoxin would trigger the coagulation cascade to produce clotting enzyme that would activate the conversion of the coagulogen to coagulin and result in the formation of a firm clot. The LAL assay (either gelclotting or end point chromogenic) is recommend by the International Organization of Standardization (ISO),14 the International Pharmacopoeia,15 and the China Food Drug Administration16 for specific detection of endotoxin in vitro for biomedical materials and devices. However, several research groups have reported that some kinds of nanomaterials brought interference to the endotoxin reaction in the LAL assay. For instance, gold colloids interfered with gel-clotting and end point chromogenic LAL in vitro test,17 and the gel-clotting LAL © 2017 American Chemical Society

Received: January 11, 2017 Accepted: March 1, 2017 Published: March 1, 2017 8450

DOI: 10.1021/acsami.7b00543 ACS Appl. Mater. Interfaces 2017, 9, 8450−8454

Letter

ACS Applied Materials & Interfaces

Figure 1. Endotoxin free O-MWCNT aqueous solution (O-MWCNT-aq) induced false positive results of gel-clotting and end point chromogenic LAL. (a) The SEM image of O-MWCNT dispersing in the solution; (b) UV−vis−NIR spectra of O-MWCN-aq at 12.5, 25, and 50 μg/mL, the inserted image showed O-MWCNT-aq with different concentrations. (c) Firm clots induced by O-MWCNT-aq, all the concentrations of OMWCNT-aq caused firm clot formation except for 1 ng/mL. (d) Image of the end point chromogenic LAL solution with the treatment of endotoxin and different concentrations of O-MWCNT-aq. (e) O-MWCNT-aq induced an increase of OD value at 545 nm in a concentration dependent manner (n = 3). (f) O-MWCNT-aq caused coagulogen conversion to coagulin analyzed by SDS-PAGE except for the concentration of 1 ng/mL.

tration of O-MWCNT (Figure 1b); the inset image shows that the blackness of solution could be diluted. Generally, in the measurement of gel-clotting LAL assay, firm clot formation is the only positive indicator of endotoxin presence. It is striking to see that endotoxin free O-MWCNTaq induced the firm clot formation rapidly when its concentration was higher than 0.01 μg/mL (Figure 1c), which strongly suggested that the O-MWCNT dispersed in water activated the LAL and provided a false indicative sign that there was endotoxin contamination. To further verify the phenomenon, we also applied the end point chromogenic LAL kit, in which the optical density (OD) at 545 nm (purple color) was measured and correlated to the concentration of endotoxin in the solution. In the test, the O-MWCNT-aq turned the LAL reagent into purple color (Figure 1d); the OD value was increased in a O-MWCNT concentration dependent manner (Figure 1e). These results indicated the O-MWCNT-aq could activate both gel-clotting and end point chromogenic LAL assays; the action way was similar to that of endotoxin. It has been well documented that the LAL activation involves sequential reactions of zymogens including limulus factor C, B,

proclotting enzyme, and coagulogen. In the complicated cascades, the final reaction is that coagulogen (21 kDa) is hydrolyzed by clotting enzyme to generate coagulin (15 kDa) that polymerizes into a solid gel. Hence we examined the protein profile of LAL assay by SDS-PAGE analysis to figure out whether coagulin appeared when the LAL reagent was encountered with O-MWCNT-aq. The endotoxin of 0.25 EU/ mL was set as a positive control that induced the conversion of coagulogen-to-coagulin (indicated by arrowheads). Strikingly, when O-MWCNT-aq was added to the gel-clotting LAL at 10, 1, 0.1, or 0.01 μg/mL, a complete conversion of coagulogen-tocoagulin was occurred (Figure 1f). It was noted that OMWCNT-aq at 0.001 μg/mL did not induce the conversion possibly because the concentration was too low, which was consistent with the result obtained from the gel-clotting LAL assay. As it is well-known that proteins are easily absorbed on the surface of carbon nanotubes,10 we hypothesized that the surface of O-MWCNT absorbed zymogen of LAL to induce the contact-activation of the zymogen. To verify this hypothesis, we first had the O-MWCNT-aq mixed with bovine serum (BS) at 8451

DOI: 10.1021/acsami.7b00543 ACS Appl. Mater. Interfaces 2017, 9, 8450−8454

Letter

ACS Applied Materials & Interfaces

MWCNT-aq was incubated with I-LAL at 37 °C for 1 h. SDSPAGE results showed that O-MWCNT-aq at each tested concentration (0.001−10 μg/mL) fail to induce the conversion of coagulogen to coagulin in I-LAL, indicating that only by activating certain enzymes in the gel-clotting LAL reagent so could O-MWCNT induce clot formation (Figure 3a). LAL clots are composed of coagulin, therefore, the other enzymes would be left in the aqueous phase when the clots formed, and the supernatants contain the excess enzymes that can induce further coagulogen conversion.20 Therefore, we supplemented O-MWCNT-aq in the gel-clotting LAL reagent to induce clot formation. After that, the clot was centrifuged and the supernatant was collected, which was then added into the I-LAL (Figure 3b). As expected, the supernatants from the clot formed by being exposed with O-MWCNT-aq (10 μg/mL or 1 μg/mL) induced complete conversion of coagulogen to coagulin, indicating a large excess amount of enzymes was left in the clot, whereas only a slight conversion was observed with O-MWCNT-aq at 0.1 μg/mL and no obvious conversion occurred when O-MWCNT-aq was at 0.01 μg/mL (Figure 3c). These results demonstrated that the coagulogen in the I-LAL reagent still had the ability to converting into coagulin, whereas O-MWCNT itself could not induce the conversion in the ILAL. The LAL assay is an important measurement that is recommended to specific detection of endotoxin and widely adopted by pharmaceutical and biomedical device industry. However, several groups reported that nanoparticles interfere to the endotoxin detection by the LAL assay,18,21 alerting that results obtained from the LAL assays in the detection of endotoxin in nanomaterials should be paid much attention. The contribution of the current investigation is to reveal the influence of endotoxin-free O-MWCNT dispersed in water upon the LAL assays and to elucidate the underlying mechanism. Particularly in this study, the O-MWCNT was treated at 250 °C for 45 min to exclude any endotoxin contamination according to the International Pharmacopoeia,15 and the endotoxin-free O-MWCNT-aq caused positive results of gel-clotting and end point chromogenic LAL assay in a concentration dependent manner. The concentration that could initiate positive response of gel-clotting and end-point chromogenic assay was a little different. In the gel-clotting assay, the initiating concentration of O-MWCNT-aq was 10 ng/mL, whereas in the chromgenic end point LAL assay, the

Table 1. DLS Analysis of O-MWCNT-aq in Bovine Serum sample O-MWCNT-aq O-MWCNT-aq+5% BS O-MWCNT-aq+10% BS O-MWCNT-aq+12.5% BS O-MWCNT-aq+15% BS 15% BS

Z-average d (nm) 128.5 153.4 152.1 149.2 129.2 17.1

± ± ± ± ± ±

1.3 1.2 2.0 1.6 2.0 0.1

zeta (mV) −32.2 −22.4 −20.6 −18.0 −15.4 −13.7

± ± ± ± ± ±

0.7 0.6 1.1 0.9 1.1 0.8

different concentrations, and then the mixture was subjected to DLS analysis. The results were given in Table 1. It was shown that the O-MWCNT-aq alone had a single population with an average hydrodynamic diameter of 128.5 nm; the zeta potential was about −32 mV. When the BS was supplemented in the OMWCNT-aq, the average dynamic size was increased, whereas the Zeta potential was decreased, which indicated that the surface of O-MWCNT was covered with BS. Next, we examined whether the BS adsorption affected the interaction between the LAL reagent and O-MWCNT using end point chromogenic LAL assay. Results showed that the OMWCNT-induced activation of LAL reagent was retarded by the BS adsorption (Figure 2a). The higher the amount of BS supplemented, the lower the OD value detected, clearly indicating that the activation of LAL occurred in the interface between the O-MWCNT and LAL zymogen, because the more BS protein adsorbed, the less of the O-MWCNT surface left to be exposed to the LAL zymogen, which resulted in lower activation of zymogen of LAL. Importantly, the conversion of coagulogen to coagulin induced by O-MWCNT-aq could be inhibited by the BS adsorption (Figure 2b). The mixture of O-MWCNT-aq with 15% BS did not induce the conversion, while O-MWCNT-aq with 10% BS inhibited half of the conversion. When BS was reduced to 5%, the conversion underwent significantly. These results confirmed that O-MWCNT induced LAL activation by the interface reactions between the two components. Next we investigated whether the other proteins in LAL assay was required in the process of O-MWCNT-aq inducing LAL activation in addition to coagulogen. It is known that LAL zymogen can be inactivated by heat treatment, whereas coagulogen can remain its ability under this condition.20 Hence we treated the gel-clotting LAL reagent at 70 °C for 20 min to prepare inactivated LAL (I-LAL). Then endotoxin or O-

Figure 2. Surface absorption of BS significantly inhibited O-MWCNT-aq inducing LAL positive results. (a) The OD (545 nm) value of chromogenic LAL induced by O-MWCNT-aq and O-MWCNT/BS. (b) Coagulogen conversion to coagulin caused by O-MWCNT-aq and O-MWCNT/BS analyzed by SDS-PAGE. 8452

DOI: 10.1021/acsami.7b00543 ACS Appl. Mater. Interfaces 2017, 9, 8450−8454

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ACS Applied Materials & Interfaces

Figure 3. LAL activation induced by O-MWCNT-aq is dependent on certain enzymes in LAL. (a) Inactivated LAL (I-LAL) treated with endotoxin or O-MWCNT-aq did not underwent the conversion of coagulogen to coagulin. (b) Procedure scheme for supernatants obtained from LAL clots induced by O-MWCNT-aq and addition to I-LAL. (c) Conversion of coagulogen to coagulin in I-LAL induced by activated supernatants.

*E-mail: [email protected].

initiating concentration was 300 ng/mL. The difference might be attributed to the formulations for the two assays. It has been recognized that nanoparticles as well as carbon nanotubes adsorb proteins rapidly and make the protein molecules assemble corona on their surface.22,23 As the treatment of oxidation and ultrasonication improved hydrophilic property of O-MWCNT largely, the adsorption LAL zymogen to the surface of O-MWCNT could be significantly increased. Therefore, it is the surface interaction of OMWCNT to LAL zymogen that initiated the activation of LAL assay and drove the clot formation, interfering to the gelclotting LAL assay significantly. Consistently, several researchers had reported that surface-contacting activation can be used to interpret the interaction of nanoparticles with zymogen activation,24 and MWCNT was preferentially absorbed factor IXa and provide a platform that enhances its enzymatic activity.25 In summary, O-MWCNT-aq activated the LAL zymogen by interface activation, causing “false” positive results. As both gelclotting and end point chromogenic LAL assays are not suitable to evaluate the endotoxin contamination in O-MWCNT-aq, substitutive methods need to be developed urgently.

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ORCID

Haiyan Xu: 0000-0002-7287-9048 Author Contributions †

M.Y. and X.N. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by Beijing Natural Science Foundation (7162128), CAMS Innovation Fund for Medical Sciences (CIFMS 2016-I2M-3-004), the PUMC Youth Fund the Fundamental Research Funds for the Central Universities (3332016136), and partially by Beijing Key Laboratory of Environmental Toxicology (2015HJDL05).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b00543. Summary of preparation procedure of MWCNT, Table S1 (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. 8453

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DOI: 10.1021/acsami.7b00543 ACS Appl. Mater. Interfaces 2017, 9, 8450−8454