Caveolin-1 and CDC42 mediated endocytosis of silica-coated iron oxide nanoparticles in HeLa cells

Abstract

Nanomedicine is a rapidly growing field in nanotechnology, which has great potential in the development of new therapies for numerous diseases. For example iron oxide nanoparticles are in clinical use already in the thermotherapy of brain cancer. Although it has been shown, that tumor cells take up these particles in vitro, little is known about the internalization routes. Understanding of the underlying uptake mechanisms would be very useful for faster and precise development of nanoparticles for clinical applications. This study aims at the identification of key proteins, which are crucial for the active uptake of iron oxide nanoparticles by HeLa cells (human cervical cancer) as a model cell line. Cells were transfected with specific siRNAs against Caveolin-1, Dynamin 2, Flotillin-1, Clathrin, PIP5Kα and CDC42. Knockdown of Caveolin-1 reduces endocytosis of superparamagnetic iron oxide nanoparticles (SPIONs) and silica-coated iron oxide nanoparticles (SCIONs) between 23 and 41%, depending on the surface characteristics of the nanoparticles and the experimental design. Knockdown of CDC42 showed a 46% decrease of the internalization of PEGylated SPIONs within 24 h incubation time. Knockdown of Dynamin 2, Flotillin-1, Clathrin and PIP5Kα caused no or only minor effects. Hence endocytosis in HeLa cells of iron oxide nanoparticles, used in this study, is mainly mediated by Caveolin-1 and CDC42. It is shown here for the first time, which proteins of the endocytotic pathway mediate the endocytosis of silica-coated iron oxide nanoparticles in HeLa cells in vitro. In future studies more experiments should be carried out with different cell lines and other well-defined nanoparticle species to elucidate possible general principles.

Keywords: CDC42; Caveolin-1; endocytosis inhibition; iron oxide nanoparticles; nanoparticle uptake.

Figure 1

Overview of well-known endocytotic pathways and the involved key proteins, target proteins inhibited in this study are marked in red (adapted from Wieffer et al. [27], sizes of membrane ruffling from Canton et al. [28]).

Figure 2

Schematic overview of SPION structure.

Figure 3

Representative X-ray films with knockdown efficiency of target proteins (red labels), ß-Actin serves as the protein loading control for comparable protein contents in the horizontal lines, control siRNA = siRNA with nonsense sequence, untreated = control cells without siRNA treatment; (a) Knockdown efficiency of Flotillin-1, Caveolin-1, Clathrin and Dynamin-2; (b) Knockdown efficiency of CDC42 and PIP5Kα.

Figure 4

Iron content of control and transfected HeLa cells in pg/cell after 24 h incubation with unmodified SPIONs; target proteins: Caveolin-1, Flotillin-1, Clathrin (iron concentration 50 µg/mL, error bars: SEM, n = 3).

Figure 5

Iron content of control and transfected HeLa cells in pg/cell after 24 h incubation with carboxylated SPIONs; target proteins: Caveolin-1, Flotillin-1, Clathrin (iron concentration 50 µg/mL, error bars: SEM, n = 3).

Figure 6

Iron content of control and transfected HeLa cells in pg/cell after 24 h incubation with PEGylated SPIONs; target proteins: Caveolin-1, Flotillin-1, Clathrin (iron concentration 50 µg/mL, error bars: SEM, n = 3).

Figure 7

Fluorescence image of Hela cells which were incubated with SCIONs (iron concentration 5 µg/mL, incubation time 4 h), blue = DAPI (nuclei), green = Transferrin Alexa Fluor^® 488 conjugate (cytosol), red = Alexa Fluor^® 555 (SCIONs); (a) Control cells without siRNA treatment; (b) Cells with knockdown of Caveolin-1.

Figure 8

Sum-fluorescence-intensity (wavelength 568) per cell of SCIONs labeled with Alexa Fluor^® 555 in transfected HeLa cells after 4 h incubation time; target proteins: Caveolin-1, Dynamin 2, Flotillin-1, Clathrin (iron concentration 5 µg/mL, n = 4).

Figure 9

Iron content of control and transfected HeLa cells in pg/cell after 24 h incubation with PEGylated SPIONs; target proteins: CDC42, PIP5Kα (iron concentration 50 µg/mL, error bars: SEM, n = 3).