Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis

Abstract

Non-phagocytic eukaryotic cells can internalize particles <1 microm in size, encompassing pathogens, liposomes for drug delivery or lipoplexes applied in gene delivery. In the present study, we have investigated the effect of particle size on the pathway of entry and subsequent intracellular fate in non-phagocytic B16 cells, using a range of fluorescent latex beads of defined sizes (50-1000 nm). Our data reveal that particles as large as 500 nm were internalized by cells via an energy-dependent process. With an increase in size (50-500 nm), cholesterol depletion increased the efficiency of inhibition of uptake. The processing of the smaller particles was significantly perturbed upon microtubule disruption, while displaying a negligible effect on that of the 500 nm beads. Inhibitor and co-localization studies revealed that the mechanism by which the beads were internalized, and their subsequent intracellular routing, was strongly dependent on particle size. Internalization of microspheres with a diameter <200 nm involved clathrin-coated pits. With increasing size, a shift to a mechanism that relied on caveolae-mediated internalization became apparent, which became the predominant pathway of entry for particles of 500 nm in size. At these conditions, delivery to the lysosomes was no longer apparent. The data indicate that the size itself of (ligand-devoid) particles can determine the pathway of entry. The clathrin-mediated pathway of endocytosis shows an upper size limit for internalization of approx. 200 nm, and kinetic parameters may determine the almost exclusive internalization of such particles along this pathway rather than via caveolae.

Biocompatibility of Thermally Hydrocarbonized Porous Silicon Nanoparticles and their Biodistribution in Rats

“…Inorganic particles below 100 nm were found to be internalized to a greater extent and to reduce viability. 25,27 However, it has also been suggested that an inflection point on the particular internalization route is apparent at a size of approximately 200 nm, 28 which is also corroborated in our study. It is for that reason that when designing a micro-or nanoparticulate drug delivery system it is advisible to avoid the thresholds that were found to have a greater impact on cell viability.…”

The endocytic pathway and therapeutic efficiency of doxorubicin conjugated cholesterol-derived polymers

“…Following inhibitor toxicity studies, confocal microscopy was employed to assess the specificity of the inhibitors in relation to the uptake of molecules known to taken up by specific endocytic routes, Human transferrin (hTF) 38 and lactosylceramide (LacCer). 39 hTF is known to be taken up via CDE while LacCer is internalized via CIE where is associated with lipid rafts. 40 Fig.…”

Intracellular trafficking of nonviral vectors

“…76 This pathway is of considerable interest in particular for molecular conjugates containing pathogen-derived trafficking peptides, since many pathogens (SV40 and cholera toxin included) are thought to use the caveolar pathway to evade degradation in the lysosomes. 77 Using different sized microspheres, Rejman et al 78 showed that molecular size can dictate whether cell entry occurs by clathrin or caveolae-mediated endocytosis. Microspheres of 200 nm in diameter or less entered cells through clathrin-coated pits by a microtubule dependent pathway.…”