Different endocytotic uptake mechanisms for nanoparticles in epithelial cells and macrophages

Abstract

SummaryPrecise knowledge regarding cellular uptake of nanoparticles is of great importance for future biomedical applications. Four different endocytotic uptake mechanisms, that is, phagocytosis, macropinocytosis, clathrin- and caveolin-mediated endocytosis, were investigated using a mouse macrophage (J774A.1) and a human alveolar epithelial type II cell line (A549). In order to deduce the involved pathway in nanoparticle uptake, selected inhibitors specific for one of the endocytotic pathways were optimized regarding concentration and incubation time in combination with fluorescently tagged marker proteins. Qualitative immunolocalization showed that J774A.1 cells highly expressed the lipid raft-related protein flotillin-1 and clathrin heavy chain, however, no caveolin-1. A549 cells expressed clathrin heavy chain and caveolin-1, but no flotillin-1 uptake-related proteins. Our data revealed an impeded uptake of 40 nm polystyrene nanoparticles by J774A.1 macrophages when actin polymerization and clathrin-coated pit formation was blocked. From this result, it is suggested that macropinocytosis and phagocytosis, as well as clathrin-mediated endocytosis, play a crucial role. The uptake of 40 nm nanoparticles in alveolar epithelial A549 cells was inhibited after depletion of cholesterol in the plasma membrane (preventing caveolin-mediated endocytosis) and inhibition of clathrin-coated vesicles (preventing clathrin-mediated endocytosis). Our data showed that a combination of several distinguishable endocytotic uptake mechanisms are involved in the uptake of 40 nm polystyrene nanoparticles in both the macrophage and epithelial cell line.

Interaction of hydroxyapatite nanoparticles with endothelial cells: internalization and inhibition of angiogenesis in vitro through the PI3K/Akt pathway

“…Three inhibitors were applied in these experiments. 24,25 Chlorpromazine (cpz) inhibits clathrin-mediated endocytosis. Methyl-β-cyclodextrin (mβcd) specifically inhibits clathrin-as well as caveolinmediated endocytosis.…”

Gold nanoparticles promote osteogenic differentiation in human adipose-derived mesenchymal stem cells through the Wnt/β-catenin signaling pathway

“…27,28 In addition, uptake mechanisms of NPs were reportedly dependent on the cell types. 29,30 In an earlier study, chitosan-conjugated AuNPs demonstrated endocytotic uptake in lung cancer cells. 25 In order to identify uptake of chitosan-conjugated AuNPs in hADMSCs, internalized chitosan-conjugated AuNPs were measured using a TEM.…”

Endocytic mechanisms and osteoinductive profile of hydroxyapatite nanoparticles in human umbilical cord Wharton’s jelly-derived mesenchymal stem cells

“…37 However, we found that cpz inhibited the uptake of m-HAP; furthermore, elegant experiments have demonstrated that NPs below 60 nm could also enter cells via clathrin-mediated endocytosis. 16,39 In summary, these findings indicate that the size of clathrin-coating vesicles might be outside the range of 150-200 nm. Given the complexity and diversity of uptake routes available in different cells, here, we explored only three main endocytic modes of these pathways.…”

“…16,17 Chlorpromazine (cpz) was used to inhibit clathrin-mediated endocytosis. Inhibitions of clathrin-as well as caveolin-mediated endocytosis in hWJ-MSCs were tested via methyl-β-cyclodextrin (mβcd).…”

“…It is furthermore well known that cells have various endocytic routes to uptake material from the extracellular milieu. 16,36 Particles with sizes above 500 nm are typically ingested via phagocytosis or macropinocytosis, while internalization of cargo below this size limit often occurs via other diverse endocytic pathways available at the cell surface, such as clathrin-mediated as well as caveolin-mediated endocytosis. 37 However, although HAP has been extensively investigated in various biomedical applications, the internalization mechanism of HANPs in MSCs still remains unknown.…”