Optimising the use of commercial LAL assays for the analysis of endotoxin contamination in metal colloids and metal oxide nanoparticles

Li, Yang; Italiani, Paola; Casals, Eudald; Tran, Ngoc; Puntes, Víctor; Boraschi, Diana

doi:10.3109/17435390.2014.948090

Cited by 52 publications

(63 citation statements)

References 37 publications

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“…However, despite these new evidences, the use of the gel clot assay is still recommended in a FDA guidance document to solve discrepancies between results from different LAL formats in industry (48). Furthermore, our results with the chromogenic LAL assay suggested that metal and metal oxide NPs may interfere with the final readout by absorbing the final dye (p-nitroaniline) and quenching the readout, leading to underestimating the endotoxin contamination (39). Therefore, Dobrovolskaia et al have declared that none of the currently available LAL formats is optimal for endotoxin assessment in ENM and suggested that at least two LAL formats with different endpoints/readouts should be used.…”

Section: Endotoxin Evaluation Methods In Nanomaterialsmentioning

confidence: 91%

“…These assays have the advantage of being totally specific for endotoxin, because β-glucan activates factor G but not factor C. Although the LAL assay can reliably detect endotoxin in soluble reagents, the physicochemical characteristics of ENM pose a significant problem of interference with both the components and the detection readouts (fluorescence, optical density) of various assays (28, 36, 39). To overcome the interference problem, the available assays need to be validated for the lack of interference by ENM with the catalytic activity of the enzyme(s), substrate cleavage, and the final readout signals (8, 39). It has been shown that the gel clot LAL assay is not accurate for testing endotoxin contamination in particles, while the chromogenic LAL assay showed higher sensitivity and no interference (46).…”

Section: Endotoxin Evaluation Methods In Nanomaterialsmentioning

confidence: 99%

“…Thus, Dobrovolskaia et al suggested to use such assays to confirm the LAL results (38). We have tested endotoxin contamination in Au, Ag, and iron oxide (Fe 3 O 4 ) NPs with the chromogenic LAL assay of Associates of Cape Cod (endpoint readout at 540 nm) and in parallel with the ECVAM-approved PBMC activation assay (IL-6 production) (39). The endotoxin contamination detected by the LAL assay was confirmed by the PBMC activation assay only for Au NPs, but not for Ag and Fe 3 O 4 NPs.…”

Section: Endotoxin Evaluation Methods In Nanomaterialsmentioning

confidence: 99%

“…This brings us to another concern, i.e., the possible interference of graphene oxide with the LAL assay. Indeed, interference has been extensively reported for many ENM (36–39), which strongly suggests the need for testing the interference for each ENM under investigation. In addition, the Lonza QCL-1000 endpoint chromogenic LAL assay with readout at 405 nm has been shown to be unsatisfactory for measuring endotoxin in metal and metal oxide (39) as well as graphene oxide ENM (40).…”

Section: Biological Effects Of Endotoxin-contaminated Nanomaterialsmentioning

confidence: 99%

“…Indeed, interference has been extensively reported for many ENM (36–39), which strongly suggests the need for testing the interference for each ENM under investigation. In addition, the Lonza QCL-1000 endpoint chromogenic LAL assay with readout at 405 nm has been shown to be unsatisfactory for measuring endotoxin in metal and metal oxide (39) as well as graphene oxide ENM (40). With all this in mind, we conclude that not only should we measure endotoxin in ENM but also we must make sure that the endotoxin detection assay is reliable and relevant to the ENM under study.…”

Section: Biological Effects Of Endotoxin-contaminated Nanomaterialsmentioning

confidence: 99%

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Endotoxin Contamination in Nanomaterials Leads to the Misinterpretation of Immunosafety Results

Fujita

Boraschi

2017

Front. Immunol.

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Given the presence of engineered nanomaterials in consumers’ products and their application in nanomedicine, nanosafety assessment is becoming increasingly important. In particular, immunosafety aspects are being actively investigated. In nanomaterial immunosafety testing strategies, it is important to consider that nanomaterials and nanoparticles are very easy to become contaminated with endotoxin, which is a widespread contaminant coming from the Gram-negative bacterial cell membrane. Because of the potent inflammatory activity of endotoxin, contaminated nanomaterials can show inflammatory/toxic effects due to endotoxin, which may mask or misidentify the real biological effects (or lack thereof) of nanomaterials. Therefore, before running immunosafety assays, either in vitro or in vivo, the presence of endotoxin in nanomaterials must be evaluated. This calls for using appropriate assays with proper controls, because many nanomaterials interfere at various levels with the commercially available endotoxin detection methods. This also underlines the need to develop robust and bespoke strategies for endotoxin evaluation in nanomaterials.

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Section: Endotoxin Evaluation Methods In Nanomaterialsmentioning

confidence: 91%

Section: Endotoxin Evaluation Methods In Nanomaterialsmentioning

confidence: 99%

Section: Endotoxin Evaluation Methods In Nanomaterialsmentioning

confidence: 99%

Section: Biological Effects Of Endotoxin-contaminated Nanomaterialsmentioning

confidence: 99%

Section: Biological Effects Of Endotoxin-contaminated Nanomaterialsmentioning

confidence: 99%

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Endotoxin Contamination in Nanomaterials Leads to the Misinterpretation of Immunosafety Results

Fujita

Boraschi

2017

Front. Immunol.

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Characterization Methods: Physical and Chemical Characterization Techniques

Borgos

2016

Pharmaceutical Nanotechnology: Innovation and Production

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Graphene and carbon nanotubes activate different cell surface receptors on macrophages before and after deactivation of endotoxins

Lahiani

Gokulan

Williams

et al. 2017

J of Applied Toxicology

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Nanomaterial synthesis and handling in a non-sterile environment can result in the final product becoming contaminated with bacterial endotoxin or lipopolysaccharides (LPB). During toxicological testing, the effects caused by endotoxin-contaminated nanomaterials can be misinterpreted in the end-point analysis (such as cytotoxicity and immune responses) and could result in erroneous conclusions. The objective of this study was twofold: (i) to test different carbonbased nanomaterials (CBNs) [pristine graphene and multi-wall carbon nanotubes (MWCNTs)] for the presence of endotoxin and develop strategies for depyrogenation, and (ii) to compare the immune response exhibited by macrophages after exposure to native CBNs versus depyrogenated CBNs. The gel-clot limulus amebocyte lysate (LAL) and chromogenic-based LAL assays were used to detect endotoxins. Results revealed that the CBNs contained greater amounts of endotoxin than are approved by major regulatory agencies (0.5 EU ml À1). Three repeated cycles of autoclaving reduced the endotoxin in the test materials. Macrophages were incubated with pyrogenated and depyrogenated pristine graphene and MWCNTs to test differences in phagocytosis, cytotoxicity, and expression of genes involved in macrophage activation. The uptake of depyrogenated CBNs was significantly reduced as compared with pyrogenated CBNs. Exposure of macrophages to depyrogenated CBNs resulted in a distinct pattern of gene expression for TLR signaling, NOD-like receptor signaling, and downstream signal transduction molecules. Furthermore, macrophages exposed to both types of CBNs showed the downregulation of TLR5 and NLRC4 inflammasomes. The results of this study reaffirm that assessment of endotoxin and other bacterial contamination is critical when evaluating the cellular toxicity of nanomaterials. Published 2017. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

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Optimising the use of commercial LAL assays for the analysis of endotoxin contamination in metal colloids and metal oxide nanoparticles

Cited by 52 publications

References 37 publications

Endotoxin Contamination in Nanomaterials Leads to the Misinterpretation of Immunosafety Results

Endotoxin Contamination in Nanomaterials Leads to the Misinterpretation of Immunosafety Results

Characterization Methods: Physical and Chemical Characterization Techniques

Graphene and carbon nanotubes activate different cell surface receptors on macrophages before and after deactivation of endotoxins

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