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.

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.

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.

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.

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 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.

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.

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.

Innate Memory Reprogramming by Gold Nanoparticles Depends on the Microbial Agents That Induce Memory.

Innate immune memory, the ability of innate cells to react in a more protective way to secondary challenges, is induced by exposure to infectious and other exogeous and endogenous agents. Engineered nanoparticles are particulate exogenous agents that, as such, could trigger an inflammatory reaction in monocytes and macrophages and could therefore be also able to induce innate memory. Here, we have evaluated the capacity of engineered gold nanoparticles (AuNPs) to induce a memory response or to modulate the memory responses induced by microbial agents. Microbial agents used were in soluble vs. particulate form (MDP and the gram-positive bacteria Staphylococcus aureus; ß-glucan and the ß-glucan-producing fungi C. albicans), and as whole microrganisms that were either killed (S. aureus, C. albicans) or viable (the gram-negative bacteria Helicobacter pylori). The memory response was assessed in vitro, by exposing human primary monocytes from 2-7 individual donors to microbial agents with or without AuNPs (primary response), then resting them for 6 days to allow return to baseline, and eventually challenging them with LPS (secondary memory response). Primary and memory responses were tested as production of the innate/inflammatory cytokine TNFα and other inflammatory and anti-inflammatory factors. While inactive on the response induced by soluble microbial stimuli (muramyl dipeptide -MDP-, ß-glucan), AuNPs partially reduced the primary response induced by whole microorganisms. AuNPs were also unable to directly induce a memory response but could modulate stimulus-induced memory in a circumscribed fashion, limited to some agents and some cytokines. Thus, the MDP-induced tolerance in terms of TNFα production was further exacerbated by co-priming with AuNPs, resulting in a less inflammatory memory response. Conversely, the H. pylori-induced tolerance was downregulated by AuNPs only relative to the anti-inflammatory cytokine IL-10, which would lead to an overall more inflammatory memory response. These effects of AuNPs may depend on a differential interaction/association between the reactive particle surfaces and the microbial components and agents, which may lead to a change in the exposure profiles. As a general observation, however, the donor-to-donor variability in memory response profiles and reactivity to AuNPs was substantial, suggesting that innate memory depends on the individual history of exposures.

SERS Sensing of Bacterial Endotoxin on Gold Nanoparticles.

Engineered gold nanoparticles (AuNPs) find application in several fields related to human activities (i.e., food and cosmetic industry or water purification) including medicine, where they are employed for diagnosis, drug delivery and cancer therapy. As for any material/reagent for human use, the safety of AuNPs needs accurate evaluation. AuNPs are prone to contamination by bacterial endotoxin (lipopolysaccharide, LPS), a potent elicitor of inflammatory responses in mammals. It is therefore important, when assessing AuNP immunosafety and immune-related effects, to discriminate between inflammatory effects intrinsic to the NPs from those caused by an undeliberate and undetected LPS contamination. Detection of LPS contamination in AuNP preparations poses different problems when using the current LPS detection assays, given the general interference of NPs, similar to other particulate agents, with the assay reagents and endpoints. This leads to time-consuming search for optimal assay conditions for every NP batch, with unpredictable results, and to the use in parallel of different assays, each with its weaknesses and unpredictability. Thus, the development of highly sensitive, quantitative and accurate assays able to detect of LPS on AuNPs is very important, in view of their medical applications. Surface-enhanced Raman spectroscopy (SERS) is a label-free, sensitive, chemical-specific, nondestructive and fast technique that can be used to directly obtain molecular fingerprint information and a quantitative analysis of LPS adsorbed on AuNPs. Within this study, we describe the use of SERS for the label-free identification and quantitative evaluation - down to few attograms - of the LPS adsorbed on the surface of 50 nm AuNPs. We thus propose SERS as an efficient tool to detect LPS on the AuNP surface, and as the basis for the development of a new sensitive and specific LPS-detection sensor based on the use of AuNPs and SERS.

Personalised Profiling of Innate Immune Memory Induced by Nano-Imaging Particles in Human Monocytes.

Engineered nanoparticles used for medical purposes must meet stringent safety criteria, which include immunosafety, i.e., the inability to activate possibly detrimental immune/inflammatory effects. Even medical nanomaterials devoid of direct immunotoxic or inflammatory effects may have an impact on human health if able to modify innate memory, which is the ability to "prime" future immune responses towards a different, possibly more detrimental reactivity. Although innate memory is usually protective, anomalous innate memory responses may be at the basis of immune pathologies. In this study, we have examined the ability of two nanomaterials commonly used for diagnostic imaging purposes, gold and iron oxide nanoparticles, to induce or modulate innate memory, using an in vitro model based on human primary monocytes. Monocytes were exposed in culture to nanoparticles alone or together with the bacterial agent LPS (priming phase/primary response), then rested for six days (extinction phase), and eventually challenged with LPS (memory/secondary response). The memory response to the LPS challenge was measured as changes in the production of inflammatory (TNFα, IL-6) and anti-inflammatory cytokines (IL-10, IL-1Ra), as compared to unprimed monocytes. The results show that both types of nanoparticles can have an effect in the induction of memory, with changes observed in the cytokine production. By comparing nanomaterials of different shapes (spherical vs. rod-shaped gold particles) and different size (17 vs. 22 nm diameter spherical iron oxide particles), it was evident that innate memory could be differentially induced and modulated depending on size, shape and chemical composition. However, the main finding was that the innate memory effect of the particles was strongly donor-dependent, with monocytes from each donor showing a distinct memory profile upon priming with the same particles, thereby making impossible to draw general conclusions on the particle effects. Thus, in order to predict the effect of imaging nanoparticles on the innate memory of patients, a personalised profiling would be required, able to take in consideration the peculiarities of the individual innate immune reactivity.

Primary and Memory Response of Human Monocytes to Vaccines: Role of Nanoparticulate Antigens in Inducing Innate Memory.

Innate immune cells such as monocytes and macrophages are activated in response to microbial and other challenges and mount an inflammatory defensive response. Exposed cells develop the so-called innate memory, which allows them to react differently to a subsequent challenge, aiming at better protection. In this study, using human primary monocytes in vitro, we have assessed the memory-inducing capacity of two antigenic molecules of Schistosoma mansoni in soluble form compared to the same molecules coupled to outer membrane vesicles of Neisseria lactamica. The results show that particulate challenges are much more efficient than soluble molecules in inducing innate memory, which is measured as the production of inflammatory and anti-inflammatory cytokines (TNFα, IL-6, IL-10). Controls run with LPS from Klebsiella pneumoniae compared to the whole bacteria show that while LPS alone has strong memory-inducing capacity, the entire bacteria are more efficient. These data suggest that microbial antigens that are unable to induce innate immune activation can nevertheless participate in innate activation and memory when in a particulate form, which is a notion that supports the use of nanoparticulate antigens in vaccination strategies for achieving adjuvant-like effects of innate activation as well as priming for improved reactivity to future challenges.

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.

Direct LC-MS/MS Analysis of Extra- and Intracellular Glycerophosphoinositol in Model Cancer Cell Lines.

Glycerophosphoinositols (GPIs) are water-soluble bioactive phospholipid derivatives of increasing interest as intracellular and paracrine mediators of eukaryotic cell functions. The most representative compound of the family is glycerophosphoinositol (GroPIns), an ubiquitous component of mammalian cells that participates in cell proliferation, cell survival and cell response to stimuli. Levels and activity of this compound vary among cell types and deciphering these functions requires accurate measurements in in vitro and in vivo models. The conventional approaches for the analysis of GroPIns pose several issues in terms of sensitivity and product resolution, especially when the product is in the extracellular milieu. Here we present an UPLC-MS study for the quantitative analysis of this lipid derivative in cells and, for the first time, culture supernatants. The method is based on a solid-phase extraction that allows for fast desalting and analyte concentration. The robustness of the procedure was tested on the simultaneous measurements of intra- and extracellular levels of GroPIns in a number of human cell lines where it has been shown that the non-transformed cells are characterized by high extracellular level of GroPIns, whereas the tumor cells tended to have higher intracellular levels.

Interaction between Macrophages and Nanoparticles: In Vitro 3D Cultures for the Realistic Assessment of Inflammatory Activation and Modulation of Innate Memory.

Understanding the modes of interaction between human monocytes/macrophages and engineered nanoparticles is the basis for assessing particle safety, in terms of activation of innate/inflammatory reactions, and their possible exploitation for medical applications. In vitro assessment of nanoparticle-macrophage interaction allows for examining the response of primary human cells, but the conventional 2D cultures do not reproduce the three-dimensional spacing of a tissue and the interaction of macrophages with the extracellular tissue matrix, conditions that shape macrophage recognition capacity and reactivity. Here, we have compared traditional 2D cultures with cultures on a 3D collagen matrix for evaluating the capacity gold nanoparticles to induce monocyte activation and subsequent innate memory in human blood monocytes in comparison to bacterial LPS. Results show that monocytes react to stimuli almost in the same way in 2D and 3D cultures in terms of production of TNFα and IL-6, but that notable differences are found when IL-8 and IL-1Ra are examined, in particular in the recall/memory response of primed cells to a second stimulation, with the 3D cultures showing cell activation and memory effects of nanoparticles better. In addition, the response variations in monocytes/macrophages from different donors point towards a personalized assessment of the nanoparticle effects on macrophage activation.

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.

Towards bio-compatible magnetic nanoparticles: Immune-related effects, in-vitro internalization, and in-vivo bio-distribution of zwitterionic ferrite nanoparticles with unexpected renal clearance.

HYPOTHESIS: Iron oxide and other ferrite nanoparticles have not yet found widespread application in the medical field since the translation process faces several big hurdles. The incomplete knowledge of the interactions between nanoparticles and living organisms is an unfavorable factor. This complex subject should be made simpler by synthesizing magnetic nanoparticles with good physical (relaxivity) and chemical (colloidal stability, anti-fouling) properties and no biological activity (no immune-related effects, minimal internalization, fast clearance). Such an innocent scaffold is the main aim of the present paper. We systematically searched for it within the class of small-to-medium size ferrite nanoparticles coated by small (zwitter)ionic ligands. Once established, it can be functionalized to achieve targeting, drug delivery, etc. and the observed biological effects will be traced back to the functional molecules only, as the nanosized scaffold is innocent. EXPERIMENTS: We synthesized nine types of magnetic nanoparticles by systematic variation of core composition, size, coating. We investigated their physico-chemical properties and interaction with serum proteins, phagocytic microglial cells, and a human model of inflammation and studied their biodistribution and clearance in healthy mice. The nanoparticles have good magnetic properties and their surface charge is determined by the preferential adsorption of anions. All nanoparticle types can be considered as immunologically safe, an indispensable pre-requisite for medical applications in humans. All but one type display low internalization by microglial BV2 cells, a process strongly affected by the nanoparticle size. Both small (3 nm) and medium size (11 nm) zwitterionic nanoparticles are in part captured by the mononuclear phagocyte system (liver and spleen) and in part rapidly (≈1 h) excreted through the urinary system of mice. FINDINGS: The latter result questions the universality of the accepted size threshold for the renal clearance of nanoparticles (5.5 nm). We suggest that it depends on the nature of the circulating particles. Renal filterability of medium-size magnetic nanoparticles is appealing because they share with small nanoparticles the decreased accumulation-related toxicity while performing better as magnetic diagnostic/therapeutic agents thanks to their larger magnetic moment. In conclusion, many of our nanoparticle types are a bio-compatible innocent scaffold with unexpectedly favorable clearance.