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

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.

[1]  J. Cavaillon,et al.  Polymyxin-B inhibition of LPS-induced interleukin-1 secretion by human monocytes is dependent upon the LPS origin. , 1986, Molecular immunology.

[2]  Christian Ehlting,et al.  The macrophage response towards LPS and its control through the p38(MAPK)-STAT3 axis. , 2012, Cellular signalling.

[3]  Sebastian Hoffmann,et al.  International validation of novel pyrogen tests based on human monocytoid cells. , 2005, Journal of immunological methods.

[4]  K. Novoselov,et al.  Exploring the Interface of Graphene and Biology , 2014, Science.

[5]  H. Krug,et al.  Oops they did it again! Carbon nanotubes hoax scientists in viability assays. , 2006, Nano letters.

[6]  N. Monteiro-Riviere,et al.  Limitations and relative utility of screening assays to assess engineered nanoparticle toxicity in a human cell line. , 2009, Toxicology and applied pharmacology.

[7]  A. Kraegeloh,et al.  Interference of silica nanoparticles with the traditional Limulus amebocyte lysate gel clot assay , 2014, Innate immunity.

[8]  Thomas Montag,et al.  Alternatives to Animal Use for the LAL-Assay , 2012 .

[9]  J. Hurley.  Endotoxemia: methods of detection and clinical correlates , 1995, Clinical microbiology reviews.

[10]  Peter Wick,et al.  Contamination of nanoparticles by endotoxin: evaluation of different test methods , 2012, Particle and Fibre Toxicology.

[11]  D. López‐Diaz,et al.  Graphene oxide thin films: influence of chemical structure and deposition methodology. , 2015, Langmuir : the ACS journal of surfaces and colloids.

[12]  Kostas Kostarelos,et al.  Purified Graphene Oxide Dispersions Lack In Vitro Cytotoxicity and In Vivo Pathogenicity , 2013, Advanced healthcare materials.

[13]  N. Bottini,et al.  Carbon nanotube-based nanocarriers: the importance of keeping it clean. , 2010, Journal of nanoscience and nanotechnology.

[14]  Rui Liu,et al.  Crucial Role of Lateral Size for Graphene Oxide in Activating Macrophages and Stimulating Pro-inflammatory Responses in Cells and Animals. , 2015, ACS nano.

[15]  Shau-ku Huang,et al.  Endotoxin contamination contributes to the in vitro cytokine-inducing activity of osteopontin preparations. , 2005, Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research.

[16]  A E Nel,et al.  Implementation of alternative test strategies for the safety assessment of engineered nanomaterials , 2013, Journal of internal medicine.

[17]  Marina A Dobrovolskaia,et al.  Choice of method for endotoxin detection depends on nanoformulation. , 2014, Nanomedicine.

[18]  Nastassja A. Lewinski,et al.  Cytotoxicity of nanoparticles. , 2008, Small.

[19]  T. Kirikae,et al.  Bacterial endotoxin: molecular relationships of structure to activity and function , 1994, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[20]  R. Baughman,et al.  Carbon Nanotubes: Present and Future Commercial Applications , 2013, Science.

[21]  Lei Wang,et al.  Graphene oxide induces toll-like receptor 4 (TLR4)-dependent necrosis in macrophages. , 2013, ACS nano.

[22]  David S. Ensor,et al.  Endotoxin contamination of engineered nanomaterials , 2010, Nanotoxicology.

[23]  Anders Hult,et al.  Stability and biocompatibility of a library of polyester dendrimers in comparison to polyamidoamine dendrimers. , 2012, Biomaterials.

[24]  Agnes B Kane,et al.  Biological interactions of graphene-family nanomaterials: an interdisciplinary review. , 2012, Chemical research in toxicology.

[25]  S. Amar,et al.  Differentiation of Monocytes to Macrophages Primes Cells for Lipopolysaccharide Stimulation via Accumulation of Cytoplasmic Nuclear Factor κB , 1999, Infection and Immunity.

[26]  Marina A Dobrovolskaia,et al.  Evaluation of nanoparticle immunotoxicity. , 2009, Nature nanotechnology.

[27]  Marina A Dobrovolskaia,et al.  Ambiguities in applying traditional Limulus amebocyte lysate tests to quantify endotoxin in nanoparticle formulations. , 2010, Nanomedicine.

[28]  R. Bishop.  Fundamentals of endotoxin structure and function. , 2005, Contributions to microbiology.

[29]  Jian Qin,et al.  The importance of an endotoxin-free environment during the production of nanoparticles used in medical applications. , 2006, Nano letters.

[30]  F. Cunha,et al.  Lipopolysaccharide Induces Inflammatory Hyperalgesia Triggering a TLR4/MyD88-Dependent Cytokine Cascade in the Mice Paw , 2014, PloS one.

[31]  J. L. García Fierro,et al.  The role of oxidative debris on graphene oxide films. , 2013, Chemphyschem : a European journal of chemical physics and physical chemistry.

[32]  Mary Gulumian,et al.  Challenges facing sterilization and depyrogenation of nanoparticles: effects on structural stability and biomedical applications. , 2014, Nanomedicine : nanotechnology, biology, and medicine.