Endotoxin contamination: An interference for nanosafety studies
Nanotechnology has undergone a rapid growth all over the world, with the production of a broad array of nanomaterials, which are increasingly entering our lives, as many are incorporated in products of common use . The effects of engineered nanomaterials on biological processes and consequently on human and environmental health are currently a topic of considerable debate. There is still a lack of knowledge concerning the safety aspects of nanomaterials . As for all novel materials, with particular regard to nanomedicines and health-related nanomaterials, immunological safety of nanomaterials should be taken with great care since the immune system takes responsibility for defending the integrity of our body and maintaining its health. However, when we consider nano-immuno safety studies, we should be aware of the fact that engineered nanomaterials are generally contaminated by bacterial endotoxin (lipopolysaccharide, LPS), a biologically active molecule with significant toxic and inflammatory effects. The presence of endotoxin can generate misleading experimental results in in vitro and in vivo assays aiming at evaluating the toxic and inflammatory effects of nanomaterials.
Endotoxin or lipopolysaccharide (LPS) is a large molecule (molecular weight: 200 to 1000 kDa) from the outer membrane of Gram-negative bacteria, which is an important component of the bacteria’s ability to cause diseases. Endotoxin consists of a polysaccharide part and a lipid part, known as lipid A, responsible for its toxicity. In mammals, including man, the toxic/inflammatory effects of LPS are mediated by the TLR4 signalling pathway. LPS binds to lipopolysaccharide-binding protein (LBP), and then engages the TLR4 receptor with the contribution of the co-receptors CD14 and MD2, thereby initiating cell activation. The activation of innate immune cells such as monocytes and macrophages by LPS through TLR4 is a major step of the defensive inflammatory reaction against bacteria . LPS-stimulated cells produce a variety of inflammatory factors, including the cytokines IL-1β, TNF-α and IL-6.
Our evaluation on a number of commercial nanomaterials showed that most of them are contaminated by endotoxin (unpublished data). A previous study performed together with the lab of Victor Puntes has shown that different batches of the same nanomaterial synthesized in the same lab show different levels of endotoxin contamination depending on the cleanliness of the synthesis procedure . This situation suggests caution in the interpretation of data showing the toxic/inflammatory effects of nanomaterials, since the unrecognized presence of endotoxin in nanomaterials may actually account for most of the observed effects or synergize with nanomaterials in inducing cell activation. Thus, the reliability of nanotoxicologcal data, either in vitro or in vivo, depends on discriminating true nano-effects from the effects of biologically active contaminants, of which bacterial endotoxin is the most common.
Endotoxin is a heat-stable ubiquitous contaminant, present in almost all the chemicals and glassware used in laboratories, unless specific precautions are taken. It is important to be aware that endotoxin cannot be eliminated by sterilisation. For obtaining an endotoxin-free nanomaterial preparation, all procedures should be performed with endotoxin-free reagents and glassware. The most efficient way of destroying endotoxin is incineration (“depyrogenation”), which is obtained for instance by dry heating at high temperatures (180°C for 3 h or 250°C for 30 min) . Depyrogenation, is very efficient in reducing endotoxin activity (5 logs reduction), however it cannot be used for treating most of the relevant nanomaterials once synthesized, because the extreme conditions will alter the physico-chemical characteristics of nanomaterials. Therefore, the best way for having low-endotoxin or endotoxin-free nanomaterials is to take precaution during the synthesis procedures .
Nanomaterials, due to the highly reactive nature of their surface, can adsorb many types of molecules from the milieu, to reduce surface free energy. Hence, unintentional surface contaminants could be introduced on the surface of nanomaterials by adsorption of air- or solution-borne species . Endotoxin is among the molecules that can attach to the nanomaterials. Endotoxin is likely to adsorb on hydrophobic surfaces through its lipid moiety, while its phosphate groups allows it to bind to positively charged surfaces. Therefore, endotoxin is in theory able to attach to all kinds of surfaces [7-8]. Already 14 years ago, R. Darkow and co-workers have tested the endotoxin binding activity by using functionalized nanoparticles . Daniel W. Karl and co-workers had also shown that endotoxin could adsorb to nanoparticles’ surface and the binding activity could be reduced with the aggregated state during the derivatisation process . Our recent study in collaboration with Victor Puntes’ lab has also shown that endotoxin could bind to gold nanoparticles’ surface in a dose-dependent fashion (unpublished data).
We thus know that endotoxin could bind to the nanomaterials surface, but what is the biological effect of this endotoxin contamination? Some researches have already addressed the effect of endotoxin on nanoparticles. David R. Cho has investigated the interaction of orthopedic wear particles with endotoxin, and found that particle-adsorbed endotoxin can markedly enhance their inflammatory activity and affect implant loosening through localised inflammation . Other data showed that co-administration of endotoxin could promote the side effects of engineered nanoparticles on lung function both in vitro and in vivo . Titanium particles, contaminated on purpose with endotoxin, could trigger inflammation in macrophages by inducing the production of inflammatory cytokines. However, these inflammatory effects seem to be quantitatively weaker than those obtained with endotoxin alone, implying that upon binding, endotoxin partly loses its activity . Another explanation may be that nanomaterials and endotoxin have opposing effects and partially neutralise each other. In this direction, David V. Pereira et al. showed that gold nanoparticles could inhibit cell activation induced by endotoxin through the TLR4-NFκB pathway, resulting in decreased inflammation and oxidative damage in rat’s uveitis . On the other hand, our recent studies show that endotoxin-treated gold nanoparticles are much more inflammatory on human monocytes compared to the clean gold nanoparticles, which have no inflammatory effect (unpublished data). The interaction of endotoxin with nanoparticles may thus vary depending on the type of nanoparticles, the type and amount of endotoxin, and the cell type or biological entities/environment under investigation. In any case, there is no doubt that presence of endotoxin may affect the impact of engineered nanoparticles on a biological system, and that such interaction needs therefore to be addressed in detail.
The biological effects of endotoxin could mask or interfere with the true biological effects of nanomaterials. Hence, reliably discriminating the endotoxin activity from the nanomaterial’s intrinsic inflammatory activity is important for nanosafety studies. The rabbit pyrogen test (RPT) and the Limulus Amoebocyte Lysate (LAL) assay are the most commonly used endotoxin detection methods that are approved by FDA and EMA and accepted by almost every country. However, due to the high cost and long turnaround time of the RPT test (and the usage of animals), nowadays RPT is only applied in combination with the LAL test for analysing the parenteral drugs during the earlier development phase. As the most popular endotoxin detection method, the LAL assay could supply fast, sensitive and specific endotoxin assessment (LAL effect can only be interfered by β-glucan, which can be inhibited by a specific buffer). In this assay, an enzyme that is derived from the blood of the horseshoe crab Limulus polyphemus clots upon exposure to endotoxin. There are three different variants of the LAL assay: gel-clot, chromogenic and turbidimetric. The LAL assay is designed for soluble molecules with certain limitations in ionic strength. Thus, the assay should be accurately validated when applied to endotoxin detection in nanomaterials. In fact, the nano-objects’ peculiar physico-chemical properties (including their optical density) could significantly interfere with the LAL readouts, thereby leading to false results . Although the European Centre for the Validation of Alternative Methods (ECVAM) published various highly sensitive and reliable bioassays for assessing pyrogens, such as the human PBMC activation assay and the human monocytes activation assay, these bioassays are not specific for endotoxin because any pyrogen (including nanomaterials) can trigger the same biological effects (usually the production of inflammatory cytokines). Thus, the pyrogen bioassays (both the two above described in vitro methods and the old RPT method) are not adequate for detecting the endotoxin contamination in nanomaterial samples. Some researchers have shown that graphene oxide and functionalized gold nanoparticles could induce TLR4-dependent necrosis or activation of macrophages in the absence of endotoxin , implying that some nanomaterials might also trigger the TLR4 signalling pathway and induce the same effects as endotoxin. These results however, as in many other cases, could be considered valid only when the presence of endotoxin or of other TLR4 ligands will be formally proven.
In summary, the ongoing nanosafety studies should take into careful consideration the presence of unwanted bioactive contaminants, of which bacterial endotoxin is the most common and abundant, when assessing nanotoxicological effects. This would avoid misinterpretation of experimental results and erroneous attribution to nanoparticles of toxic effects that may be entirely due to contaminants. To this end, not only the biologist/toxicologist should be aware of the danger and appropriately design and perform his/her experiments, but chemists should re-design synthesis processes so as to avoid contamination.
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