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Glucan (from Alcaligenes faecalis) is a polymer composed of β-1,3-linked glucose residues, and it has been addressed in different medical fields, namely in nanotechnology, as a vaccine or a drug delivery system. However, due to their small size, nanomaterials may present new risks and uncertainties. Thus, this work aims to describe the production of glucan nanoparticles (NPs) with two different sizes, and to evaluate the influence of the NPs size on immunotoxicity. Results showed that, immediately after production, glucan NPs presented average sizes of 129.7 ± 2.5 and 355.4 ± 41.0 nm. Glucan NPs of 130 nm presented greater ability to decrease human peripheral blood mononuclear cells and macrophage viability and to induce reactive oxygen species production than glucan NPs of 355 nm. Both NP sizes caused hemolysis and induced a higher metabolic activity in lymphocytes, although the concentration required to observe such effect was lower for the 130 nm glucan NPs. Regarding pro-inflammatory cytokines, only the larger glucan NPs (355 nm) were able to induce the secretion of IL-6 and TNF-α, probably due to their recognition by dectin-1. This higher immunomodulatory effect of the larger NPs was also observed in its ability to stimulate the production of nitric oxide (NO) and IL-1β. On the contrary, a small amount of Glu 130 NPs inhibited NO production. In conclusion, on the safe-by-design of glucan NPs, the size of the particles should be an important critical quality attribute to guarantee the safety and effectiveness of the nanomedicine.

Even though nanotechnology has revolutionized the biomedical research, a plethora of studies debate the nanoparticles safety. In order to contribute to these studies, we evaluated the cytotoxic and epigenetic effects of maghemite nanoparticles covered with citric acid on human submandibular gland cells. Objective: This work objective was to evaluate the cytotoxic effects and epigenetic alterations induced in human salivary gland cells after treatment with maghemite nanoparticles covered with citric acid. Methods: For that, human submandibular gland cells were cultured and treated with nanoparticles for 24 or 48 hours. To assess cytotoxicity we used lactate dehydrogenase, a general oxidative stress indicator assay and microscopy. Epigenetic status was detected by colorimetric assays and the results were confirmed by quantitative polymerase chain reaction. Results: No cytotoxic effects were detected on cells exposed to up 3.0 mgFemL-1 for 48 hours, although cytoplasmic vacuoles formation was detected by light microscopy analyses. An increased generation of reactive oxygen species in cells exposed to nanoparticles was evidenced and iron clusters accumulated in the cytoplasm of treated cells. Global DNA methylation and histones H3 and H4 acetylation were also altered in response to nanoparticles exposure, thus suggesting a reprogramming of the epigenome. Transcripts accumulation analyses showed that genes related to iron metabolism and oxidative stress were upregulated, while the gene related to epigenetic reprogramming presented reduced transcript accumulation after treatment. Conclusion: We concluded that maghemite nanoparticles covered with citric acid exposure provoked several biological responses without impairment of human submandibular gland cells viability. This is the first report on the epigenetic effects of maghemite nanoparticles on this cell lineage.

Poly-epsilon-caprolactone (PCL) is a biodegradable polyester that has FDA and CE approval as a medical device. Nonetheless, the lack of toxicity exhibited by the polymer cannot be extrapolated to its nanomaterial conformation. Despite PCL-based NPs being widely studied in the biomedical field for their advantages as controlled drug delivery systems, little data describe PCL NPs' toxicity, particularly immunotoxicity. This work assessed different PCL-based delivery systems intended for protein delivery regarding their immunotoxicity and hemocompatibility. Two different molecular weight PCL polymers were used, as well as blends with chitosan and glucan. Results showed that the presence of NaOH during the production of PCL2 NPs and PCL2/glucan NPs induced PCL alkali hydrolysis, generating more reactive groups (carboxyl and hydroxyl) that contributed to an increased toxicity of the NPs (higher reduction in peripheral blood mononuclear cell viability and lower hemocompatibility). PCL2/glucan NPs showed an anti-inflammatory activity characterized by the inhibition of LPS stimulated nitric oxide (NO) and TNF-alpha. In conclusion, generalizations among different PCL NP delivery systems must be avoided, and immunotoxicity assessments should be performed in the early stage of product development to increase the clinical success of the nanomedicine. European Regional Development Fund (ERDF) through the COMPETE 2020-Operational Programme for Competitiveness and Internationalization Portuguese national funds via FCT-Fundacao para a Ciencia e Tecnologia [POCI-01-0145-FEDER-030331, POCI-01-0145-FEDER-032610-PTDC/MEC-DER/32610/2017, UIDB/04565/2020] info:eu-repo/semantics/publishedVersion

Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis, the mobilization of stored triacylglycerol. This work provides an important basis for generating reproducible and detailed data on the hydrolytic and transacylation activities of ATGL. We generated full-length and C-terminally truncated ATGL variants fused with various affinity tags and analyzed their expression in different hosts, namely E. coli, the insect cell line Sf9, and the mammalian cell line human embryonic kidney 293T. Based on this screen, we expressed a fusion protein of ATGL covering residues M1-D288 flanked with N-terminal and C-terminal purification tags. Using these fusions, we identified key steps in expression and purification protocols, including production in the E. coli strain ArcticExpress (DE3) and removal of copurified chaperones. The resulting purified ATGL variant demonstrated improved lipolytic activity compared with previously published data, and it could be stimulated by the coactivator protein comparative gene identification-58 and inhibited by the protein G0/G1 switch protein 2. Shock freezing and storage did not affect the basal activity but reduced coactivation of ATGL by comparative gene identification 58. In vitro, the truncated ATGL variant demonstrated acyl-CoA–independent transacylation activity when diacylglycerol was offered as substrate, resulting in the formation of fatty acid as well as triacylglycerol and monoacylglycerol. However, the ATGL variant showed neither hydrolytic activity nor transacylation activity upon offering of monoacylglycerol as substrate. To understand the role of ATGL in different physiological contexts, it is critical for future studies to identify all its different functions and to determine under what conditions these activities occur.

The use of particulate adjuvants offers an interesting possibility to enhance and modulate the immune responses elicited by vaccines. Aluminium salts have been extensively used as vaccine adjuvants, but they lack the capacity to induce a strong cellular and mucosal immune response. Taking this into consideration, in this study we designed a new antigen delivery system combining aluminium salts with chitosan. Chitosan-aluminium nanoparticles (CH-Al NPs) exhibited a mean diameter of 280nm and a positive surface charge. The newly developed CH-Al NPs are more stable at physiological environment than classical CH NPs, showing no cytotoxic effects and revealing potential as a delivery system for a wide range of model antigens. In vivo studies showed that mice immunized with hepatitis B surface antigen (HBsAg)-containing CH NPs display high anti-HBsAg IgG titers in the serum, as well as the highest antigen-specific IgG on vaginal washes. Furthermore, in contrast to mice receiving antigen alone, mice immunized with the particulate adjuvant were able to elicit IgG2c antibody titers and exhibited higher antigen-specific IFN-γ levels in splenocytes. In conclusion, we established that CH-Al NPs, combining two immunostimulants to enhance both humoral and cellular immune responses, are a safe and promising system for antigen delivery. Our findings point towards their potential in future vaccination approaches.

Airborne environmental particles (EP) more commonly referred as particulate matter (PM) are an illustrative marker of air pollution that is associated with adverse effects on human health. Considering, PM is a complex mixture, not only in terms of its chemical composition, but also in the range of particle size, it is difficult to identify which attribute contributes more for the toxicity. Currently, there is no report about the immunotoxicological effects caused by PM with reduced content of heavy metals. This study intends to address this gap and provides a detailed characterization and immunotoxicity evaluation of PM collected in an urban area with heavy traffic congestion. Environmental particles were separated by different sizes though a sucrose gradient. This method allowed to achieve 4 sized fractions: EP f 15 % with a mean diameter of 284 nm ± 1.86 nm, EP f 25 % with a mean diameter of 461 nm ± 1.72 nm, EP f 35 % with a mean diameter of 1845 nm ± 251 nm and EP f 45 % with a mean diameter of 2204 nm ± 310 nm. Only the fractions with the smallest sizes (EP f 15 % and EP f 25 %) were subsequently studied. The chemical composition of both fractions was not substantially different, and the dominant elements were C, O, Ca and K. Only EP f 25 % showed to have a small amount of Fe. Therefore, the heavy metal elements were eliminated through centrifugation. Essentially, we found that the EP f 15 % was more cytotoxic in RAW 264.7 cells than EP f 25 %, which indicates the smaller size as the motive for the higher toxicity. In addition, both fractions of EP presented a good internalization in macrophages after 2 h exposure and induced the production of reactive oxygen species in a concentration-dependent manner. Moreover, EP f 15 % and EP f 25 % led to a strong secretion of proinflammatory cytokines (TNF-α and IL-6) in human peripheral blood mononuclear cells (hPBMCs) in the 3 concentrations tested. The inflammatory response observed was independent of the presence of heavy metals and endotoxins, since these last were suppressed by using polymyxin B sulfate. This report emphasizes the importance of an adequate physicochemical characterization and adequate controls in the experiments to achieve a right interpretation of the biological effects caused by PM.