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WIREs Nanomed Nanobiotechnol
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Endotoxin contamination of engineered nanomaterials: Overcoming the hurdles associated with endotoxin testing

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Abstract Nanomaterials are highly susceptible to endotoxin contamination due their large surface‐to‐volume ratios and endotoxins propensity to associate readily to hydrophobic and cationic surfaces. Additionally, the stability of endotoxin ensures it cannot be removed efficiently through conventional sterilization techniques such as autoclaving and ionizing radiation. In recent times, the true significance of this hurdle has come to light with multiple reports from the United States Nanotechnology Characterization Laboratory, in particular, along with our own experiences of endotoxin testing from multiple Horizon 2020‐funded projects which highlight the importance of this issue for the clinical translation of nanomaterials. Herein, we provide an overview on the topic of endotoxin contamination of nanomaterials intended for biomedical applications. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine
Endotoxin assessment of a leading IONP. Following interference in the chromogenic assay, the UV–Vis absorbance spectra of the nanomaterial showed that the IONP absorbed highly at the wavelengths used in the assay (0.91 AU at 405 nm). With wavelengths used in the diazo chromogenic assay, however, it absorbed considerably less (0.23 AU at 550 nm). Based on this, the diazo chromogenic assay was successfully used to overcome the interference and determine the endotoxin contamination in the nanomaterial. This concentration was deemed too high to fulfill the requirements of a medical device and so the nanomaterials constituents were tested to determine the source of contamination. The polymer coating around the nanomaterial was found to have a considerable amount of endotoxin, whereas the iron oxide core contributed less than half this concentration, and the diluent had levels of endotoxin that were undetectable by the assay. The transmission electron microscopy image scale bar represents 100 μm
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Overcoming inhibitions with the chromogenic assay. Various iron oxide nanoparticles (IONP) were found to strongly inhibit the chromogenic assay with an absorption endpoint of 405 nm. Switching the endpoint to 550 nm via the addition of the diazo reagent successfully overcame this interference in each case. Characteristics of each nanoparticle (core, coating, functional group, and size) were provided by the suppliers. Red lines on graph depict the acceptable margins for spike‐recovery (50–200%). Abbreviations: PEG, polyethylene glycol
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Endotoxin binding to cationic and hydrophobic surfaces on nanomaterials. (a) Nanomaterials with cationic surfaces are particularly susceptible to endotoxin binding due to their negatively charged phosphate groups (illustrated with red bars). Endotoxin can also form micelles in aqueous environments at high concentrations due to their hydrophobic lipids and hydrophilic polysaccharides, which too can interact electrostatically via their available phosphate groups. (b) The lipid A structure on endotoxin can hydrophobically bind to lipophilic surfaces on nanomaterials. Endotoxin contamination is not exclusive to cationic and hydrophobic nanomaterials. Endotoxin can be incorporated into nanomaterials at any point during synthesis or handling through environmental contamination or through the use of reagents or other chemical components that are not endotoxin‐free
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Endotoxin signaling and downstream effects. Endotoxin introduced into the circulation by nanomaterials can activate the TLR4 signaling and stimulate the immune system. LBP can bind directly to endotoxin and facilitate its association to serum or membrane bound CD14, which, in turn, transfers endotoxin to the TLR4/MD2 receptor complex. Here, TIRAP‐MYD88 adaptor protein signaling is activated at the plasma membrane, resulting in pro‐inflammatory cytokine expression. Additionally, the TLR4/MD2 receptor complex can become endocytosed which subsequently activates TRAM‐TRIF signaling, leading to Type I interferon expression
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Schematic representation of endotoxin derived from Escherichia coli O111:B4. Endotoxin derives from the outer membrane of gram‐negative bacteria. The bioactive lipid A region is embedded in the cell membrane, acting as an anchor for the polysaccharide backbone. The O‐antigen region consists of repeating oligosaccharide units which varies in structure between bacterial species. The red bars on endotoxin depict negatively charged phosphate groups. The endotoxin structure provided herein is an illustration of the chemical structure of E. coli 0111:B4 (Magalhães et al., 2007)
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Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine
Therapeutic Approaches and Drug Discovery > Emerging Technologies

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