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This study aims to highlight the impact of physicochemical properties on the behaviour of nanopharmaceuticals and how much carrier structure and physiochemical characteristics weigh on the effects of a formulation. For this purpose, two commercially available nanosimilar formulations of Doxil and their respective carriers were compared as a case study. Although the two formulations were "similar", we detected different toxicological effects (profiles) in terms of in vitro toxicity and immunological responses at the level of cytokines release and complement activation (iC3b fragment), that could be correlated with the differences in the physicochemical properties of the formulations. Shedding light on nanosimilar key quality attributes of liposome-based materials and the need for an accurate characterization, including investigation of the immunological effects, is of fundamental importance considering their great potential as delivery system for drugs, genes, or vaccines and the growing market demand.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Della Camera, Madej, Ferretti, La Spina, Li, Corteggio, Heinzl, Swartzwelter, Sipos, Gioria, Ponti, Boraschi and Italiani.)

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.

The capacity of engineered nanoparticles to activate cells of the innate immune system, in particular monocytes and macrophages, is considered at the basis of their toxic/inflammatory effects. It is, however, evident that even nanoparticles that do not directly induce inflammatory activation, and are therefore considered as safe, can nevertheless induce epigenetic modifications and affect metabolic pathways in monocytes and macrophages. Since epigenetic and metabolic changes are the main mechanisms of innate memory, we had previously proposed that nanoparticles can induce/modulate innate memory, that is, have the ability of shaping the secondary response to inflammatory challenges. In light of new data, it is now possible to support the original hypothesis and show that different types of nanoparticles can both directly induce innate memory, priming macrophages for a more potent response to subsequent stimuli, and modulate bacteria-induced memory by attenuating the priming-induced enhancement. This evidence raises two important issues. First, in addition to overt toxic/inflammatory effects, we should consider evaluating the capacity to induce innate memory and the related epigenetic and metabolic changes in the immunosafety assessment of nanomaterials, since modulation of innate memory may be at the basis of long-term unwanted immunological effects. The other important consideration is that this capacity of nanomaterials could open a new avenue in immunomodulation and the possibility of using engineered nanomaterials for improving immune responses to vaccines and resistance to infections, and modulate anomalous immune/inflammatory reactions in chronic inflammatory diseases, autoimmunity, and a range of other immune-related pathologies., (Copyright © 2020 Italiani, Della Camera and Boraschi.)

Monocytes and macrophages have a central role in all phases of an inflammatory reaction. To understanding the regulation of monocyte activation during a physiological or pathological inflammation, we propose two in vitro models that recapitulate the different phases of the reaction (recruitment, initiation, development, and resolution vs. persistence of inflammation), based on human primary blood monocytes exposed to sequential modifications of microenvironmental conditions. These models exclusively describe the functional development of blood-derived monocytes that first enter an inflammatory site. All reaction phases were profiled by RNA-Seq, and the two models were validated by studying the modulation of IL-1 family members. Genes were differentially modulated, and distinct clusters were identified during the various phases of inflammation. Pathway analysis revealed that both models were enriched in pathways involved in innate immune activation. We observe that monocytes acquire an M1-like profile during early inflammation, and switch to a deactivated M2-like profile during both the resolving and persistent phases. However, during persistent inflammation they partially maintain an M1 profile, although they lose the ability to produce inflammatory cytokines compared to M1 cells. The production of IL-1 family molecules by ELISA reflected the transcriptomic profiles in the distinct phases of the two inflammatory reactions. Based on the results, we hypothesize that persistence of inflammatory stimuli cannot maintain the M1 activated phenotype of incoming monocytes for long, suggesting that the persistent presence of M1 cells and effects in a chronically inflamed tissue is mainly due to activation of newly incoming cells. Moreover, being IL-1 family molecules mainly expressed and secreted by monocytes during the early stages of the inflammatory response (within 4-14 h), and the rate of their production decreasing during the late phase of both resolving and persistent inflammation, we suppose that IL-1 factors are key regulators of the acute defensive innate inflammatory reaction that precedes establishment of longer-term adaptive immunity, and are mainly related to the presence of recently recruited blood monocytes. The well-described role of IL-1 family cytokines and receptors in chronic inflammation is therefore most likely dependent on the continuous influx of blood monocytes into a chronically inflamed site., (Copyright © 2020 Italiani, Mosca, Della Camera, Melillo, Migliorini, Milanesi and Boraschi.)

The immune defensive mechanisms active in the solitary ascidian Ciona robusta include phagocytic and encapsulating activity, largely brought about by phagocytic cells within the haemocyte population, the presence of complement components, which have been molecularly and functionally identified, and expression of a number of immune-related genes and pathways, identified by genome-based homology with vertebrate counterparts. Since C. robusta only displays highly conserved innate immune mechanisms, being devoid of an adaptive immune system, this organism is an excellent model for studying the features of innate memory, i.e., the capacity of the innate immune system to re-programming its responsiveness to potentially dangerous agents upon repeated exposure. In this study, we have developed an in vivo model for assessing the establishment and molecular/functional features of innate memory, by sequentially exposing C. robusta to a priming stimulus (microbial molecules), followed by a period of resting to return to basal conditions, and a challenge with microbial agents in homologous or cross-stimulation. The endpoints of immune activation were a functional activity (phagocytosis) and the molecular profiles of immune-related gene expression. The results show that exposure of C. robusta to microbial agents induces a reaction that primes animals for developing a different (expectedly more protective) response to subsequent challenges, showing the effective establishment of an immune memory. This immune memory relies on the modulation of a number of different mechanisms, some of which are priming-specific, others that are challenge-specific, and others that are non-specific, i.e., are common to all priming/challenge combinations (e.g., up-regulation of the Tnf and Lbp genes). Memory-dependent expression of the humoral immunity-related gene C3ar inversely correlates with memory-dependent variations of phagocytic rate, suggesting that complement activation and phagocytosis are alternative defensive mechanisms in C. robusta . Conversely, memory-dependent expression of the cellular immunity-related gene Cd36 directly correlates with variations of phagocytic rate, suggesting a direct involvement of this gene in the functional regulation of phagocytosis.