Endotoxin contamination of nanoparticle formulations: A concern in vaccine adjuvant mechanistic studies.

The increasing awareness of endotoxin contamination has raised important questions during the study of the mechanism of action of the vaccine adjuvants. The endotoxins or lipopolysaccharides (LPS) can contaminate vaccine formulations contributing to result misinterpretations of the in vitro and in vivo studies. In this short communication, we considered the suitability of the Limulus amebocyte lysate (LAL) assay to quantify chitosan (Chit) nanoparticle (NP) endotoxin contamination to use them in a comparative in vitro immunotoxicology study using both LPS-free (LF) and non-LF Chit NPs. It was shown that chit NPs had a masking effect on endotoxin levels, hampering a reliable conclusion about the effect of their contamination. Neither non-LF nor LF Chit NPs induced the production of ROS in RAW 264.7 cells or IL-6 and TNF-α in PBMCs. The lack of effect of non-LF NPs was not expected and likely due to the NPs masking effect, more evident for higher deacetylation degree Chit. Overall, to prevent questionable results, nanomaterials should be produced under endotoxin-free conditions.

Chitosan Nanoparticles: Shedding Light on Immunotoxicity and Hemocompatibility.

Nanoparticles (NPs) assumed an important role in the area of drug delivery. Despite the number of studies including NPs are growing over the last years, their side effects on the immune system are often ignored or omitted. One of the most studied polymers in the nano based drug delivery system field is chitosan (Chit). In the scientific literature, although the physicochemical properties [molecular weight (MW) or deacetylation degree (DDA)] of the chitosan, endotoxin contamination and appropriate testing controls are rarely reported, they can strongly influence immunotoxicity results. The present work aimed to study the immunotoxicity of NPs produced with different DDA and MW Chit polymers and to benchmark it against the polymer itself. Chit NPs were prepared based on the ionic gelation of Chit with sodium tripolyphosphate (TPP). This method allowed the production of two different NPs: Chit 80% NPs (80% DDA) and Chit 93% NPs (93% DDA). In general, we found greater reduction in cell viability induced by Chit NPs than the respective Chit polymers when tested in vitro using human peripheral blood monocytes (PBMCs) or RAW 264.7 cell line. In addition, Chit 80% NPs were more cytotoxic for PBMCs, increased reactive oxygen species (ROS) production (above 156 µg/mL) in the RAW 264.7 cell line and interfered with the intrinsic pathway of coagulation (at 1 mg/mL) when compared to Chit 93% NPs. On the other hand, only Chit 93% NPs induced platelet aggregation (at 2 mg/mL). Although Chit NPs and Chit polymers did not stimulate the nitric oxide (NO) production in RAW 264.7 cells, they induced a decrease in lipopolysaccharide (LPS)-induced NO production at all tested concentrations. None of Chit NPs and polymers caused hemolysis, nor induced PBMCs to secrete TNF-α and IL-6 cytokines. From the obtained results we concluded that the DDA of the Chit polymer and the size of Chit NPs influence the in vitro immunotoxicity results. As the NPs are more cytotoxic than the corresponding polymers, one should be careful in the extrapolation of trends from the polymer to the NPs, and in the comparisons among delivery systems prepared with different DDA chitosans.

Safe-by-Design of Glucan Nanoparticles: Size Matters When Assessing the Immunotoxicity.

Glucan (from Alcaligenes faecalis) is a polymer composed of ß-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-1ß. 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.

Airborne environmental fine particles induce intense inflammatory response regardless of the absence of heavy metal elements.

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.

Abelardo Duarte: medicina, virtude e trabalho / Aberlado Duarte: medicine, virtue and work

Faz um enfoque sobre a vida e trajetória profissional do médico alagoano Aberlado Duarte.