Perfluorocarbon nanoparticle uptake under physiological and inflammatory conditions depends on particle size and envelope

Inflammatory hot spots, as in infarcted myocardium, can be visualized with ¹⁹F magnetic resonance imaging (MRI) after i. v. application of ¹⁹F tracers. However, little is known about cellular uptake mechanisms of nanoparticles carrying perfluorocarbons as ¹⁹F tracers, e. g. perfluorocarbon nanoemusion (PFC) or Poly-lactic-coglycolic acid nanoparticles (PLGA), under healthy and inflammatory conditions. This study systematically investigates cellular uptake mechanisms of small and large PFCs (sPFC, bPFC) and of PLGAs by cultured cells, and by primary immune cells isolated from peripheral blood and organs under healthy, or nonsterile (subcutaneous Matrigel/lipopolysaccharide (LPS)) and sterile (Myocardial infarction (MI)) inflammation in male C57BL/6J mice.

PFC (83 and 299 nm) or PLGA (226 nm) uptake in vivo and in vitro was assessed by flow cytometry. Analysed cells were leukocytes isolated from animals with inflammation and sham controls, or cultured macrophages and leukocytes isolated from healthy animals incubated with different particle concentrations, at 37 or 4 °C, without plasma, with FcR block, chlorpromazine to block clathrin-mediated endocytosis or cytochalasin D to block phagocytosis. Additionally, PFCs or PLGAs were injected i. v., followed by repeated ¹H/¹⁹F MRI of blood, liver, and spleen over 24 h, and bone marrow, lymph nodes and the inflammatory foci after 24 h.

We found a proportionally stronger increase in nanoparticle uptake by macrophage cell lines for higher concentrations of PLGAs compared with PFCs, and a remarkable strong initial uptake of high concentrations of sPFC only, by peripheral blood neutrophils. In vivo uptake by immune cell subtypes in different organs trended to vary between nanoparticle types, and immune cell subtypes with the highest “percentage of positive cells to total ¹⁹F signal” ratio in peripheral organs differed between nanoparticle types. Incubating cells at low temperature, revealed that uptake of bPFCs by cell lines and of PLGAs by monocytes and neutrophils only mildly depended on active uptake mechanisms. Nanoparticle uptake depended on the presence of plasma components, especially for sPFCs and, to a significantly lower degree, PLGAs in neutrophils and B cells. Blocking Fc receptors temporarily reduced sPFC uptake by T cells. Nanoparticle-uptake, especially of sPFCs was reduced upon blocking clathrin-mediated endocytosis, while sPFC uptake was increased for monocytes and neutrophils, but reduced for T cells upon blocking phagocytosis. Ex vivo peripheral blood, and even more so Matrigel immune cell sPFC uptake was increased at early time-points in the presence of a subcutaneous Matrigel/LPS plug, both for monocytes, and even more so for neutrophils, while PLGAs only at later time-points showed an increased uptake by monocytes derived from the Matrigel plug. Early uptake of sPFCs by peripheral blood neutrophils isolated from animals 48 h after MI was increased.

This study shows that cellular uptake depends on nanoparticle type under both healthy and inflammatory conditions, and that uptake mechanisms depend on nanoparticle size and envelope. Remarkable was a strong initial uptake of sPFCs by neutrophils under both sterile and non-sterile inflammatory conditions, and that plasma components had the strongest impact on uptake of sPFCs, while PLGA uptake was mainly independent of the investigated uptake mechanisms.