Wide application of nano- and microparticles requires new effective approaches for evaluation of interaction of micro- and nanoparticles with body tissues. Human macrophage system has developed some mechanisms for recognition, engulfment and elimination by oxidation/digestion of crystals and nanoparticles. However, this approach does not work in the case of hydrophobic, inert or sharp edged particles, usually resulting in some damaging of engulfing macrophages and stimulating recruitment of neutrophils. The latter was shown to form neutrophil extracellular traps (NETs) being able to surround, sequester and eventually isolate nanoparticles, preventing their further contact with the body. Tracking of nano- and microparticles is often challenging, because it requires radioactive labeling or the use of other complicated methods of detection. Air pouch model is an artificial cavity, usually induced on the back of experimental mice by injection of sterile air. In some time it becomes widely populated with different immune cells and was effectively used for many years with the aim to study different biological processes like pharmacokinetics, angiogenesis or cell migration. We have recently demonstrated the use of the air pouch model for analysis the sequestration of nanodiamonds, polystyrene nanoparticles or carbon nanoparticles.
In the current work we described the methodological part related to the use of the air pouch model for evaluation of immune cells’ ability to sequester injected foreign particles. As examples we used aluminium oxide nanowires, aluminium hydroxide and graphene nanoparticles.
The formers are often injected to the body during vaccination process as adjuvants, and were recently shown to be entrapped by NETs at the place of injection, while the latter is a very resistant example of carbon pollutants, which are becoming a serious danger during forest fires, caused by climate changes and other factors.
On the basis of histopathological studies, the formation of air pouch was found to be ideal on the 6th day based on mechanical barrier and blood vessels, and the infiltration of immune cells was optimal 24 hours after the injection of the tested sample. Thus, the analysis of nanoparticle interaction can be done within 7 days. Significant numbers of eosinophils, monocytes and mastocytes, involved in the development of the inflammatory process, were detected inside the air pouch. Neutrophils in the air pouch were forming NETs. We observed entrapment of both γ-Al2O3 NWs and Al(OH)3 compounds and the formation of aggregated NETs around them. However, micro-capillary damage and edema were significantly less visible for γ-Al2O3 NWs than for commercial alum. At the same time the inert carbon nanomaterial graphene oxide was not sequestered or phagocytosed by any of the cell types infiltrating the air pouch cavity and 24 hours post-injection was still filling all space of air pouch cavity.
The proposed approach can be effectively utilized to study interactions of immune cells with other particulate nanomatter.
Keywords: air pouch model, adjuvants, micro- and nanoparticles, neutrophil extracellular traps
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