Heterogeneity of Lipopolysaccharide as Source of Variability in Bioassays and LPS-Binding Proteins as Remedy.

Lipopolysaccharide (LPS), also referred to as endotoxin, is the major component of Gram-negative bacteria's outer cell wall. It is one of the main types of pathogen-associated molecular patterns (PAMPs) that are known to elicit severe immune reactions in the event of a pathogen trespassing the epithelial barrier and reaching the bloodstream. Associated symptoms include fever and septic shock, which in severe cases, might even lead to death. Thus, the detection of LPS in medical devices and injectable pharmaceuticals is of utmost importance. However, the term LPS does not describe one single molecule but a diverse class of molecules sharing one common feature: their characteristic chemical structure. Each bacterial species has its own pool of LPS molecules varying in their chemical composition and enabling the aggregation into different supramolecular structures upon release from the bacterial cell wall. As this heterogeneity has consequences for bioassays, we aim to examine the great variability of LPS molecules and their potential to form various supramolecular structures. Furthermore, we describe current LPS quantification methods and the LPS-dependent inflammatory pathway and show how LPS heterogeneity can affect them. With the intent of overcoming these challenges and moving towards a universal approach for targeting LPS, we review current studies concerning LPS-specific binders. Finally, we give perspectives for LPS research and the use of LPS-binding molecules.

Induction of Innate Memory in Human Monocytes Exposed to Mixtures of Bacterial Agents and Nanoparticles.

We assessed whether concomitant exposure of human monocytes to bacterial agents and different engineered nanoparticles can affect the induction of protective innate memory, an immune mechanism that affords better resistance to diverse threatening challenges. Monocytes were exposed in vitro to nanoparticles of different chemical nature, shape and size either alone or admixed with LPS, and cell activation was assessed in terms of production of inflammatory (TNFα, IL-6) and anti-inflammatory cytokines (IL-10, IL-1Ra). After return to baseline conditions, cells were re-challenged with LPS and their secondary "memory" response measured. Results show that nanoparticles alone are essentially unable to generate memory, while LPS induced a tolerance memory response (less inflammatory cytokines, equal or increased anti-inflammatory cytokines). LPS-induced tolerance was not significantly affected by the presence of nanoparticles during the memory generation phase, although with substantial donor-to-donor variability. This suggests that, despite the overall lack of significant effects on LPS-induced innate memory, nanoparticles may have donor-specific effects. Thus, future nanosafety assessment and nanotherapeutic strategies will need a personalized approach in order to ensure both the safety and efficacy of nano medical compounds for individual patients.

The family of the interleukin-1 receptors.

The extracellular forms of the IL-1 cytokines are active through binding to specific receptors on the surface of target cells. IL-1 ligands bind to the extracellular portion of their ligand-binding receptor chain. For signaling to take place, a non-binding accessory chain is recruited into a heterotrimeric complex. The intracellular approximation of the Toll-IL-1-receptor (TIR) domains of the 2 receptor chains is the event that initiates signaling. The family of IL-1 receptors (IL-1R) includes 10 structurally related members, and the distantly related soluble protein IL-18BP that acts as inhibitor of the cytokine IL-18. Over the years the receptors of the IL-1 family have been known with many different names, with significant confusion. Thus, we will use here a recently proposed unifying nomenclature. The family includes several ligand-binding chains (IL-1R1, IL-1R2, IL-1R4, IL-1R5, and IL-1R6), 2 types of accessory chains (IL-1R3, IL-1R7), molecules that act as inhibitors of signaling (IL-1R2, IL-1R8, IL-18BP), and 2 orphan receptors (IL-1R9, IL-1R10). In this review, we will examine how the receptors of the IL-1 family regulate the inflammatory and anti-inflammatory functions of the IL-1 cytokines and are, more at large, involved in modulating defensive and pathological innate immunity and inflammation. Regulation of the IL-1/IL-1R system in the brain will be also described, as an example of the peculiarities of organ-specific modulation of inflammation.

Nanoparticles and innate immunity: new perspectives on host defence.

The innate immune system provides the first line of defence against foreign microbes and particulate materials. Engineered nanoparticles can interact with the immune system in many different ways. Nanoparticles may thus elicit inflammation with engagement of neutrophils, macrophages and other effector cells; however, it is important to distinguish between acute and chronic inflammation in order to identify the potential hazards of nanoparticles for human health. Nanoparticles may also interact with and become internalised by dendritic cells, key antigen-presenting cells of the immune system, where a better understanding of these processes could pave the way for improved vaccination strategies. Nanoparticle characteristics such as size, shape and deformability also influence nanoparticle uptake by a plethora of immune cells and subsequent immune responses. Furthermore, the corona of adsorbed biomolecules on nanoparticle surfaces should not be neglected. Complement activation represents a special case of regulated and dynamic corona formation on nanoparticles with important implications in clearance and safety. Additionally, the inadvertent binding of bacterial lipopolysaccharide to nanoparticles is important to consider as this may skew the outcome and interpretation of immunotoxicological studies. Here, we discuss nanoparticle interactions with different cell types and soluble mediators belonging to the innate immune system.

A Step-by-Step Approach to Improve Clinical Translation of Liposome-Based Nanomaterials, a Focus on Innate Immune and Inflammatory Responses.

This study aims to provide guidelines to design and perform a robust and reliable physical-chemical characterization of liposome-based nanomaterials, and to support method development with a specific focus on their inflammation-inducing potential. Out of eight differently functionalized liposomes selected as "case-studies", three passed the physical-chemical characterization ( in terms of size-distribution, homogeneity and stability) and the screening for bacterial contamination (sterility and apyrogenicity). Although all three were non-cytotoxic when tested in vitro, they showed a different capacity to activate human blood cells. HSPC/CHOL-coated liposomes elicited the production of several inflammation-related cytokines, while DPPC/CHOL- or DSPC/CHOL-functionalized liposomes did not. This work underlines the need for accurate characterization at multiple levels and the use of reliable in vitro methods, in order to obtain a realistic assessment of liposome-induced human inflammatory response, as a fundamental requirement of nanosafety regulations.

Addressing Nanomaterial Immunosafety by Evaluating Innate Immunity across Living Species.

The interaction of a living organism with external foreign agents is a central issue for its survival and adaptation to the environment. Nanosafety should be considered within this perspective, and it should be examined that how different organisms interact with engineered nanomaterials (NM) by either mounting a defensive response or by physiologically adapting to them. Herein, the interaction of NM with one of the major biological systems deputed to recognition of and response to foreign challenges, i.e., the immune system, is specifically addressed. The main focus is innate immunity, the only type of immunity in plants, invertebrates, and lower vertebrates, and that coexists with adaptive immunity in higher vertebrates. Because of their presence in the majority of eukaryotic living organisms, innate immune responses can be viewed in a comparative context. In the majority of cases, the interaction of NM with living organisms results in innate immune reactions that eliminate the possible danger with mechanisms that do not lead to damage. While in some cases such interaction may lead to pathological consequences, in some other cases beneficial effects can be identified.

The Impact of Nanoparticles on Innate Immune Activation by Live Bacteria.

The innate immune system evolved to detect and react against potential dangers such as bacteria, viruses, and environmental particles. The advent of modern technology has exposed innate immune cells, such as monocytes, macrophages, and dendritic cells, to a relatively novel type of particulate matter, i.e., engineered nanoparticles. Nanoparticles are not inherently pathogenic, and yet cases have been described in which specific nanoparticle types can either induce innate/inflammatory responses or modulate the activity of activated innate cells. Many of these studies rely upon activation by agonists of toll-like receptors, such as lipopolysaccharide or peptidoglycan, instead of the more realistic stimulation by whole live organisms. In this review we examine and discuss the effects of nanoparticles on innate immune cells activated by live bacteria. We focus in particular on how nanoparticles may interfere with bacterial processes in the context of innate activation, and confine our scope to the effects due to particles themselves, rather than to molecules adsorbed on the particle surface. Finally, we examine the long-lasting consequences of coexposure to nanoparticles and bacteria, in terms of potential microbiome alterations and innate immune memory, and address nanoparticle-based vaccine strategies against bacterial infection.

Circulating levels of IL-1 family cytokines and receptors in Alzheimer's disease: new markers of disease progression?

BACKGROUND: Although the mechanisms underlying AD neurodegeneration are not fully understood, it is now recognised that inflammation could play a crucial role in the initiation and progression of AD neurodegeneration. A neuro-inflammatory network, based on the anomalous activation of microglial cells, includes the production of a number of inflammatory cytokines both locally and systemically. These may serve as diagnostic markers or therapeutic targets for AD neurodegeneration. METHODS: We have measured the levels of the inflammation-related cytokines and receptors of the IL-1 family in serum of subjects with AD, compared to mild cognitive impairment (MCI), subjective memory complaints (SMC), and normal healthy subjects (NHS). Using a custom-made multiplex ELISA array, we examined ten factors of the IL-1 family, the inflammation-related cytokines IL-1α, IL-1ß, IL-18, and IL-33, the natural inhibitors IL-1Ra and IL-18BP, and the soluble receptors sIL-1R1, sIL-1R2, sIL-1R3, and sIL-1R4. RESULTS: The inflammatory cytokines IL-1α and IL-1ß, their antagonist IL-1Ra, and their soluble receptor sIL-1R1 were increased in AD. The decoy IL-1 receptor sIL-1R2 was only increased in MCI. IL-33 and its soluble receptor sIL-1R4 were also significantly higher in AD. The soluble form of the accessory receptor for both IL-1 and IL-33 receptor complexes, sIL-1R3, was increased in SMC and even more in AD. Total IL-18 levels were unchanged, whereas the inhibitor IL-18BP was significantly reduced in MCI and SMC, and highly increased in AD. The levels of free IL-18 were significantly higher in MCI. CONCLUSIONS: AD is characterised by a significant alteration in the circulating levels of the cytokines and receptors of the IL-1 family. The elevation of sIL-1R4 in AD is in agreement with findings in other diseases and can be considered a marker of ongoing inflammation. Increased levels of IL-1Ra, sIL-1R1, sIL-1R4, and IL-18BP distinguished AD from MCI and SMC, and from other inflammatory diseases. Importantly, sIL-1R1, sIL-1R3, sIL-1R4, and IL-18BP negatively correlated with cognitive impairment. A significant elevation of circulating sIL-1R2 and free IL-18, not present in SMC, is characteristic of MCI and disappears in AD, making them additional interesting markers for evaluating progression from MCI to AD.

IL-1 family cytokines and receptors in IgG4-related disease.

BACKGROUND/AIM: The IgG4-related disease (IgG4-RD) is a fibroinflammatory condition that can affect almost any organ, often associated with eosinophilia and increased levels of IgE and IgG4. Overexpression in tissues of Th2-related cytokines but also of IFN-γ has been reported. Given the major role of Il-1 family cytokines in inducing and regulating inflammation, and the paucity of data so far available in IgG-RD, we performed a comprehensive analysis of IL-18, related IL-1 family cytokines and soluble receptors in these patients. PATIENTS AND METHODS: Fifteen patients fulfilling the criteria for the diagnosis of IgG4-RD and 80 blood donors as control were recruited. Cytokines of the IL-1 family (IL-1α, IL-1ß, IL-33, IL-18), soluble receptors (sIL-1R1, sIL-1R2, sIL-1R3, ST2/sIL-1R4) and antagonists (IL-1Ra, IL-18 binding protein -IL-18BP-) were measured in sera by multiarray ELISA assay. Free IL-18 was calculated as the amount of IL-18 not inhibited by IL-18BP. RESULTS: Half of the patients had a multiorgan disease, mainly affecting retroperitoneum, lymph nodes and pancreas. sIL-1R1 (p=0.0001), sIL-1R2 (p=0.0024), ST2/sIL-1R4 (p=0.002) were significantly increased in IgG4-RD sera compared with healthy controls; sIL-R3 was significantly lower in patients vs controls (p=0,0006). CONCLUSIONS: The increased levels of the soluble forms of the two IL-1 receptors IL-1R1 and IL-1R2 suggest the need to dampen IL-1-mediated inflammation at the tissue level. Elevated circulating ST2/sIL-1R4 levels may represent the marker of an ongoing protective mechanism, but their contribution to organ damage cannot be excluded. On the whole, the data suggest a tight control of IL-1 family cytokines signalling in IgG4-RD.

Evaluating the levels of interleukin-1 family cytokines in sporadic amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease leading to the death of affected individuals within years. The involvement of inflammation in the pathogenesis of neurodegenerative diseases, including ALS, is increasingly recognized but still not well understood. The aim of this study is to evaluate the levels of inflammation-related IL-1 family cytokines (IL-1ß, IL-18, IL-33, IL-37) and their endogenous inhibitors (IL-1Ra, sIL-1R2, IL-18BP, sIL-1R4) in patients with sporadic ALS (sALS), METHODS: Sera were collected from 144 patients (125 patients were characterized by disease form, duration, and disability, using the revised ALS functional rating scale (ALSFRS-R) and from 40 matched controls. Cerebrospinal fluid (CSF) was collected from 54 patients with sALS and 65 patients with other non-infectious non-oncogenic diseases as controls. Cytokines and inhibitors were measured by commercial ELISA. RESULTS: Among the IL-1 family cytokines tested total IL-18, its endogenous inhibitor IL-18BP, and the active form of the cytokine (free IL-18) were significantly higher in the sALS sera than in controls. No correlation between these soluble mediators and different clinical forms of sALS or the clinical setting of the disease was found. IL-18BP was the only mediator detectable in the CSF of patients. CONCLUSIONS: Among the IL-1 family cytokines, only IL-18 correlates with this disease and may therefore have a pathological role in sALS. The increase of total IL-18 suggests the activation of IL-18-cleaving inflammasome. Whether IL-18 upregulation in circulation of sALS patients is a consequence of inflammation or one of the causes of the pathology still needs to be addressed.

Innate and germline immune memory: specificity and heritability of the ancient immune mechanisms for adaptation and survival.

The immune memory is one of the defensive strategies developed by both unicellular and multicellular organisms for ensuring their integrity and functionality. While the immune memory of the vertebrate adaptive immune system (based on somatic recombination) is antigen-specific, encompassing the generation of memory T and B cells that only recognize/react to a specific antigen epitope, the capacity of vertebrate innate cells to remember past events is a mostly non-specific mechanism of adaptation. This "innate memory" can be considered as germline-encoded because its effector tools (such as innate receptors) do not need somatic recombination for being active. Also, in several organisms the memory-related information is integrated in the genome of germline cells and can be transmitted to the progeny for several generations, but it can also be erased depending on the environmental conditions. Overall, depending on the organism, its environment and its living habits, innate immune memory appears to be a mechanism for achieving better protection and survival against repeated exposure to microbes/stressful agents present in the same environment or occurring in the same anatomical district, able to adapt to changes in the environmental cues. The anatomical and functional complexity of the organism and its lifespan drive the generation of different immune memory mechanisms, for optimal adaptation to changes in the living/environmental conditions. The concept of innate immunity being non-specific needs to be revisited, as a wealth of evidence suggests a significant degree of specificity both in the primary immune reaction and in the ensuing memory-like responses. This is clearly evident in invertebrate metazoans, in which distinct scenarios can be observed, with both non-specific (immune enhancement) or specific (immune priming) memory-like responses. In the case of mammals, there is evidence that some degree of specificity can be attained in different situations, for instance as organ-specific protection rather than microorganism-specific reaction. Thus, depending on the challenges and conditions, innate memory can be non-specific or specific, can be integrated in the germline and transmitted to the progeny or be short-lived, thereby representing an exceptionally plastic mechanism of defensive adaptation for ensuring individual and species survival.

Boosting antigen-specific T cell activation with lipid-stabilized protein nanoaggregates.

Vaccines based on protein antigens have numerous advantages over inactivated pathogens, including easier manufacturing and improved safety. However, purified antigens are weakly immunogenic, as they lack the spatial organization and the associated 'danger signals' of the pathogen. Formulating vaccines as nanoparticles enhances the recognition by antigen presenting cells, boosting the cell-mediated immune response. This study describes a nano-precipitation method to obtain stable protein nanoaggregates with uniform size distribution without using covalent cross-linkers. Nanoaggregates were formed via microfluidic mixing of ovalbumin (OVA) and lipids in the presence of high methanol concentrations. A purification protocol was set up to separate the nanoaggregates from OVA and liposomes, obtained as byproducts of the mixing. The nanoaggregates were characterized in terms of morphology, ζ-potential and protein content, and their interaction with immune cells was assessed in vitro. Antigen-specific T cell activation was over 6-fold higher for nanoaggregates compared to OVA, due in part to the enhanced uptake by immune cells. Lastly, a two-dose immunization with nanoaggregates in mice induced a significant increase in OVA-specific CD8+ T splenocytes compared to soluble OVA. Overall, this work presents for the first time the microfluidic production of lipid-stabilized protein nanoaggregates and provides a proof-of-concept of their potential for vaccination.

Cause or consequence? The role of IL-1 family cytokines and receptors in neuroinflammatory and neurodegenerative diseases.

Cytokines and receptors of the IL-1 family are key mediators in innate immune and inflammatory reactions in physiological defensive conditions, but are also significantly involved in immune-mediated inflammatory diseases. Here, we will address the role of cytokines of the IL-1 superfamily and their receptors in neuroinflammatory and neurodegenerative diseases, in particular Multiple Sclerosis and Alzheimer's disease. Notably, several members of the IL-1 family are present in the brain as tissue-specific splice variants. Attention will be devoted to understanding whether these molecules are involved in the disease onset or are effectors of the downstream degenerative events. We will focus on the balance between the inflammatory cytokines IL-1ß and IL-18 and inhibitory cytokines and receptors, in view of future therapeutic approaches.

Environmental stress and nanoplastics' effects on Ciona robusta: regulation of immune/stress-related genes and induction of innate memory in pharynx and gut.

In addition to circulating haemocytes, the immune system of the solitary ascidian Ciona robusta relies on two organs, the pharynx and the gut, and encompasses a wide array of immune and stress-related genes. How the pharynx and the gut of C. robusta react and adapt to environmental stress was assessed upon short or long exposure to hypoxia/starvation in the absence or in the presence of polystyrene nanoplastics. We show that the immune response to stress is very different between the two organs, suggesting an organ-specific immune adaptation to the environmental changes. Notably, the presence of nanoplastics appears to alter the gene modulation induced by hypoxia/starvation in both organs, resulting in a partial increase in gene up-regulation in the pharynx and a less evident response to stress in the gut. We have also assessed whether the hypoxia/starvation stress could induce innate memory, measured as gene expression in response to a subsequent challenge with the bacterial agent LPS. Exposure to stress one week before challenge induced a substantial change in the response to LPS, with a general decrease of gene expression in the pharynx and a strong increase in the gut. Co-exposure with nanoplastics only partially modulated the stress-induced memory response to LPS, without substantially changing the stress-dependent gene expression profile in either organ. Overall, the presence of nanoplastics in the marine environment seems able to decrease the immune response of C. robusta to stressful conditions, hypothetically implying a reduced capacity to adapt to environmental changes, but only partially affects the stress-dependent induction of innate memory and subsequent responses to infectious challenges.

The SARS-CoV-2 Nucleoprotein Induces Innate Memory in Human Monocytes.

The interaction of SARS-CoV-2 with the human immune system is at the basis of the positive or negative outcome of the infection. Monocytes and macrophages, which are major innate immune/inflammatory effector cells, are not directly infected by SARS-CoV-2, however they can react to the virus and mount a strong reaction. Whether this first interaction and reaction may bias innate reactivity to re-challenge, a phenomenon known as innate memory, is currently unexplored and may be part of the long-term sequelae of COVID-19. Here, we have tested the capacity of SARS-CoV-2 and some of its proteins to induce innate memory in human monocytes in vitro. Our preliminary results show that the Spike protein subunits S1 and S2 and the entire heat-inactivated virus have no substantial effect. Conversely, monocytes pre-exposed to the nucleocapsid N protein react to subsequent viral or bacterial challenges with an increased production of anti-inflammatory IL-1Ra, a response profile suggesting a milder response to new infections.

Methodological Approaches To Assess Innate Immunity and Innate Memory in Marine Invertebrates and Humans.

Assessing the impact of drugs and contaminants on immune responses requires methodological approaches able to represent real-life conditions and predict long-term effects. Innate immunity/inflammation is the evolutionarily most widespread and conserved defensive mechanism in living organisms, and therefore we will focus here on immunotoxicological methods that specifically target such processes. By exploiting the conserved mechanisms of innate immunity, we have examined the most representative immunotoxicity methodological approaches across living species, to identify common features and human proxy models/assays. Three marine invertebrate organisms are examined in comparison with humans, i.e., bivalve molluscs, tunicates and sea urchins. In vivo and in vitro approaches are compared, highlighting common mechanisms and species-specific endpoints, to be applied in predictive human and environmental immunotoxicity assessment. Emphasis is given to the 3R principle of Replacement, Refinement and Reduction of Animals in Research and to the application of the ARRIVE guidelines on reporting animal research, in order to strengthen the quality and usability of immunotoxicology research data.

Problems and challenges in the development and validation of human cell-based assays to determine nanoparticle-induced immunomodulatory effects.

BACKGROUND: With the increasing use of nanomaterials, the need for methods and assays to examine their immunosafety is becoming urgent, in particular for nanomaterials that are deliberately administered to human subjects (as in the case of nanomedicines). To obtain reliable results, standardised in vitro immunotoxicological tests should be used to determine the effects of engineered nanoparticles on human immune responses. However, before assays can be standardised, it is important that suitable methods are established and validated. RESULTS: In a collaborative work between European laboratories, existing immunological and toxicological in vitro assays were tested and compared for their suitability to test effects of nanoparticles on immune responses. The prototypical nanoparticles used were metal (oxide) particles, either custom-generated by wet synthesis or commercially available as powders. Several problems and challenges were encountered during assay validation, ranging from particle agglomeration in biological media and optical interference with assay systems, to chemical immunotoxicity of solvents and contamination with endotoxin. CONCLUSION: The problems that were encountered in the immunological assay systems used in this study, such as chemical or endotoxin contamination and optical interference caused by the dense material, significantly affected the data obtained. These problems have to be solved to enable the development of reliable assays for the assessment of nano-immunosafety.

In Vitro and In Vivo Models to Assess the Immune-Related Effects of Nanomaterials.

The immunological safety of drugs, nanomaterials and contaminants is a central point in the regulatory evaluation and safety monitoring of working and public places and of the environment. In fact, anomalies in immune responses may cause diseases and hamper the physical and functional integrity of living organisms, from plants to human beings. In the case of nanomaterials, many experimental models are used for assessing their immunosafety, some of which have been adopted by regulatory bodies. All of them, however, suffer from shortcomings and approximations, and may be inaccurate in representing real-life responses, thereby leading to incomplete, incorrect or even misleading predictions. Here, we review the advantages and disadvantages of current nanoimmunosafety models, comparing in vivo vs. in vitro models and examining the use of animal vs. human cells, primary vs. transformed cells, complex multicellular and 3D models, organoids and organs-on-chip, in view of implementing a reliable and personalized nanoimmunosafety testing. The general conclusion is that the choice of testing models is key for obtaining reliable predictive information, and therefore special attention should be devoted to selecting the most relevant and realistic suite of models in order to generate relevant information that can allow for safer-by-design nanotechnological developments.

The Interactions between Nanoparticles and the Innate Immune System from a Nanotechnologist Perspective.

The immune system contributes to maintaining the body's functional integrity through its two main functions: recognizing and destroying foreign external agents (invading microorganisms) and identifying and eliminating senescent cells and damaged or abnormal endogenous entities (such as cellular debris or misfolded/degraded proteins). Accordingly, the immune system can detect molecular and cellular structures with a spatial resolution of a few nm, which allows for detecting molecular patterns expressed in a great variety of pathogens, including viral and bacterial proteins and bacterial nucleic acid sequences. Such patterns are also expressed in abnormal cells. In this context, it is expected that nanostructured materials in the size range of proteins, protein aggregates, and viruses with different molecular coatings can engage in a sophisticated interaction with the immune system. Nanoparticles can be recognized or passed undetected by the immune system. Once detected, they can be tolerated or induce defensive (inflammatory) or anti-inflammatory responses. This paper describes the different modes of interaction between nanoparticles, especially inorganic nanoparticles, and the immune system, especially the innate immune system. This perspective should help to propose a set of selection rules for nanosafety-by-design and medical nanoparticle design.

Interaction of nanoparticles with endotoxin Importance in nanosafety testing and exploitation for endotoxin binding.

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.