Multiple Internalization Pathways of Polyelectrolyte Multilayer Capsules into Mammalian Cells

Abstract

Polyelectrolyte multilayer (PEM) capsules are carrier vehicles with great potential for biomedical applications. With the future aim of designing biocompatible, effective therapeutic delivery systems (e.g., for cancer), the pathway of internalization (uptake and fate) of PEM capsules was investigated. In particular the following experiments were performed: (i) the study of capsule co-localization with established endocytic markers, (ii) switching-off endocytotic pathways with pharmaceutical/chemical inhibitors, and (iii) characterization and quantification of capsule uptake with confocal and electron microscopy. As result, capsules co-localized with lipid rafts and with phagolysosomes, but not with other endocytic vesicles. Chemical interference of endocytosis with chemical blockers indicated that PEM capsules enter the investigated cell lines through a mechanism slightly sensitive to electrostatic interactions, independent of clathrin and caveolae, and strongly dependent on cholesterol-rich domains and organelle acidification. Microscopic characterization of cells during capsule uptake showed the formation of phagocytic cups (vesicles) to engulf the capsules, an increased number of mitochondria, and a final localization in the perinuclear cytoplasma. Combining all these indicators we conclude that PEM capsule internalization in general occurs as a combination of different sequential mechanisms. Initially, an adsorptive mechanism due to strong electrostatic interactions governs the stabilization of the capsules at the cell surface. Membrane ruffling and filopodia extensions are responsible for capsule engulfing through the formation of a phagocytic cup. Co-localization with lipid raft domains activates the cell to initiate a lipid-raft-mediated macropinocytosis. Internalization vesicles are very acidic and co-localize only with phagolysosome markers, excluding caveolin-mediated pathways and indicating that upon phagocytosis the capsules are sorted to heterophagolysosomes.

In vivo fate of free and encapsulated iron oxide nanoparticles after injection of labelled stem cells

“…The difference in Fe uptake is unlikely to be due to electrostatic interactions, 25,33 as both formulations were highly positively charged (Fig. SI.1 c & d).…”

“…Depending upon the cell line/type and size/dimensions of capsules, the number of micro capsules phagocytosed by cells cannot exceed a limit of 2 -15 capsules/cell. 33 So for low labelling concentrations a higher Fe uptake is achieved by using encapsulated particles. Higher degrees of labelling have been reported with high labelling concentrations of free particles.…”

“…It has been observed that a final layer of positively charged polymers results in increased uptake of capsules. 33 For long term cellular imaging the walls of the capsules can be made of non-biodegradable materials. 34 We and others have shown that most cells die after injection.…”

Intracellular delivery and sensing based on polyelectrolyte multilayer capsules

“…In contrast, small capsules in the size of nanometers are of interest for in vivo applications, where large capsules cannot be used as they may block capillaries. The cellular uptake is a statistical process that in vitro occurs in almost all mammalian cells, both immortal cell lines and primary cells [26][27][28]. It is triggered by phagocytosis and lipid-rafts-mediated macropinocytosis and the final location of the capsules is in the heterophagolysosomes of the cells [27].…”

Mechanism of hepatic targeting via oral administration of DSPE–PEG–cholic acid-modified nanoliposomes

“…Accordingly, protection from endosomal and lysosomal transport systems by caveolae-and clathrin-mediated endocytosis enabled improved entry of DOX⋅HCl into nuclei, and thus, promised improved antitumor effects. 33,34 To ensure receptor and ligand binding and achieve an active targeting effect at the target site in orally administered receptor-mediated active targeting systems, the nanoparticles must maintain their integrity during absorption by the gastrointestinal tract to ensure that the contained drug does not leak before reaching the target site. Through the energy transfer from an excited fluorophore (donor) to a nearby light-absorbing molecule (acceptor), FRET is dependent on the proximity of both fluorescent molecules, within a range of 1-10 nm of each other.…”