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The interaction between engineered nanoparticles and the bacterial lipopolysaccharide, or endotoxin, is an event that warrants attention. Endotoxin is one of the most potent stimulators of inflammation and immune reactions in human beings, and is a very common contaminant in research labs. In nanotoxicology and nanomedicine, the presence of endotoxin on the nanoparticle surface affects their biological properties leading to misinterpretation of results. This review discusses the importance of detecting the endotoxin contamination on nanoparticles, focusing on the current method of endotoxin detection and their suitability for nanoparticulate materials. Conversely, the capacity of nanoparticles to bind endotoxin can be enhanced by functionalization with endotoxin-capturing molecules, opening the way to the development of novel endotoxin detection assays., The authors were supported by the EU H2020 grants ENDONANO [GA 812661] and PANDORA [GA 671881], the IG grant [21420] of the Associazione Italiana Ricerca sul Cancro (AIRC), the project NeON [ARS01_00769] of the Program PRIN, and the projects CIRO- Campania Infrastructure for Research in Oncology and PLATT of the Programma Operativo Regionale of Regione Campania [POR FERS 2014/2020].

Removal of endotoxins from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of the bacterial expression systems widely used to manufacture therapeutic proteins. In this study we investigated various parameters affecting anion exchange chromatography to selectively remove endotoxins from therapeutic proteins. NY-ESO-1, Melan-A, and SSX-2 are different recombinant proteins used in this study, all of them are cancer antigens currently developed as potential immunotherapeutic agents. We found that by using a commercially available Q XL resin in a flow-through mode, endotoxin could be effectively removed from these proteins while maintaining very acceptable protein yields. The ratio of resin volume to endotoxin load was analyzed to determine the endotoxin binding capacity of the resin. In our hands at least 900,000 endotoxin units (EU) could be loaded per ml of Q XL resin. Solution conductivity could be increased to 20 mS/cm to minimize protein loss by weakening protein-resin attraction, and pH could be increased to enhance endotoxin removal by weakening endotoxin-protein attraction. Endotoxin levels were ultimately decreased to below 0.5 EU per microg of protein, an over 2000-fold reduction in this single step. A successful scale-up of these processes in which column volume was increased 100-fold was performed under cGMP conditions with over 80% protein recovery.

Deletion homologues of the cloned Factor C cDNAs from the horseshoe crab Carcinoscorpius rotundicauda were engineered to express in Saccharomyces cerevisiae under the regulation of a galactose-inducible promoter. Expression cassettes were constructed in the vectors: pEMBLyex4 and YEpsec1 to direct, respectively, the intracellular expression, and the secretion of the protein into the culture medium using a heterologous signal sequence. The effect of insert size on the efficiency of expression and the functionality of the resulting recombinant Factor C (rFC) were studied by creating expression constructs bearing various deletion and/or hybrid fragments of Factor C. Removal of the long 5' UTR from the Factor C cDNA improved expression of the rFC. 3' Deletions of up to 84%, or internal deletions of 65% of the Factor C cDNA resulted in either the lack of detectable amounts of Factor C or loss of immunoreactivity. Depending on the construct, full length or partial rFC-related proteins were correspondingly expressed intracellularly, regardless of the vector. The rFC partitioned with the insoluble cell fraction, was solubilised with either SDS or Triton X-100, and found to be immunoreactive. The rFCs were functionally active, being able to bind Gram-negative bacterial endotoxin, provided critical regions of the endotoxin-binding domain were preserved.

The endotoxin inactivating action of plasma was evaluated in 62 patients with cirrhosis and 10 healthy subjects. Endotoxin from E. coli 0111:B4 was added to each plasma sample to a final concentration of 250 pg/ml and the percentage loss of endotoxin activity by incubation (37 degrees C for 1 h) was calculated as the endotoxin inactivating rate. The plasma endotoxin inactivating rate in cirrhotics was significantly greater than that in healthy subjects, although patients with Child C cirrhosis and marked hyperbilirubinemia had a significantly lower endotoxin inactivating rate than other cirrhotics. The plasma endotoxin inactivating rate was positively correlated to serum HDL-cholesterol levels. In patients with Child A and Child B cirrhosis, the endotoxin inactivating rate was positively correlated to the endotoxin binding capacity of plasma albumin. The present results support the assumption that the plasma of cirrhotics has a high endotoxin inactivating capacity. Its decrease may augment endotoxicity in patients with advanced liver cirrhosis.

This study was undertaken to determine the extent of in vitro penetration of E. coli endotoxin into the root cementum of periodontally healthy and diseased teeth. Freshly extracted teeth were washed in distilled water, scaled and divided into two groups of 16 teeth each. Nine diseased and five healthy teeth in the first group were immersed in various concentrations of E. coli endotoxin for 2 to 12 weeks. They were then prepared for indirect immunofluorescence examination after treatment with anti-endotoxin antibody and rhodamine conjugated secondary antibody. Teeth in the second group were prepared for autoradiographic examination by immersing nine diseased and five healthy teeth in tritium labelled E. coli endotoxin for 2 to 12 weeks. The latter technique also allowed for semi-quantitative study of the depth of endotoxin penetration by creating facets on the root at various depths after endotoxin exposure. This technique was also used to investigate the strength of endotoxin binding to the tooth surface by brushing for 1 minute and re-examining the tooth for the presence of endotoxin. Controls included periodontally diseased and healthy teeth. Results of the study showed that (1) endotoxin adheres to the tooth surface without penetration into the root cementum of either periodontally healthy or diseased teeth, and (2) the binding of the endotoxin to the root surface appears to be weak.

Previous studies have suggested that immaturity of the gastrointestinal barrier of young animals may lead to increased uptake of potentially toxic macromolecules. To further extend these observations, we have studied the binding of endotoxin (E. Coli 026: B6) to immature (crypt) and mature (villus) small intestinal epithelial cells of 2 week old and adult rats. Endotoxin was radiolabeled with 51Cr, and its concentration measured using a hexose assay. Cells were obtained from the small intestine in a gradient from villus to crypt using the modified method of Weiser. Binding was studied by incubating endotoxin with the cells. The endotoxin binding was found to be concentration-dependent and saturable, suggesting receptor, not nonspecific, binding. Endotoxin was incubated with immature crypt cells and mature villus cells of 2 week old rats at concentrations ranging from 5-100 μg of endotoxin/ml and at a cell protein concentration of .2 mg/ml. Approximately two times as much endotoxin bound to crypt as to villus cells. Preliminary binding experiments with epithelial cells from adult rats also show differences in endotoxin binding to crypt versus villus cells. These studies suggest that the surface of the immature cell has different binding properties for this potential toxin. The increased binding may place the immature host at a greater risk for enteric infection.