Caveolin-1 and CDC42 mediated endocytosis of silica-coated

iron oxide nanoparticles in HeLa cells

Nils Bohmer*1 and Andreas Jordan1,2

Full Research Paper

Address:
1Project Biomedical Nanotechnologies, Charit University Medicine,
13353 Berlin, Germany and 2MagForce Nanotechnologies AG, 12489
Berlin, Germany
Email:
Nils Bohmer* - nilsbohmer@gmx.de

Open Access
Beilstein J. Nanotechnol. 2015, 6, 167176.
doi:10.3762/bjnano.6.16
Received: 19 March 2014
Accepted: 18 December 2014
Published: 14 January 2015
This article is part of the Thematic Series "Biological responses to NPs".

* Corresponding author
Guest Editor: R. Zellner
Keywords:
Caveolin-1; CDC42; endocytosis inhibition; iron oxide nanoparticles;
nanoparticle uptake

2015 Bohmer and Jordan; licensee Beilstein-Institut.

License and terms: see end of document.

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, PIP5K 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 PIP5K 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.

Introduction
Nanotechnology is expected to be a very powerful technique for
the treatment of various diseases in the 21st century. Today
nanomedicine has spread in many different subareas, which are
working highly interdisciplinary on the development of new
therapy concepts [1].

One of the most important fields is the early detection and treatment of cancer. Therefore many strategies and specific nanoparticle constructs have been explored in recent years [2-4],
although only few of them have already made their way into
practice [5]. Iron oxide nanoparticles are of special interest

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because of their magnetic properties, which make them suitable

for clinical applications. Nowadays they are already in use in
magnetic resonance imaging [6-8] and the thermotherapy of
tumors [9-11]. Also for the investigation of applications in
theragnostic and drug delivery iron oxide nanoparticles are
promising tools for the future [12-18].
Despite the commercial use of iron oxide nanoparticles and
their diversified development for future applications in
nanomedicine, little is known about the way they are internalized by tumor cells or cells of other origins. By the use of
microscopic techniques previous studies showed, that iron oxide
nanoparticles often appear in vesicular structures within the
cytosol of cells in vitro [19-24], which indicates an active,
energy dependent uptake via endocytosis. In a post mortem
study of glioma patients, who had received thermotherapy with
aminosilane coated iron oxide nanoparticles in a phase-II study,
nanoparticles were mostly found in macrophages than in the
cancer cells themselves [25]. In the respective study it was not
crucial for successful treatment that the nanoparticles were
specifically taken up by the tumor cells, because they were
injected directly into the tumor and had no further payload attached to the surface. But for drug delivery applications and
intravenous injections it would be very useful to understand,
how cancer cells internalize iron oxide nanoparticles and which
pathways are involved. Insights in the principles of nanoparticle endocytosis would be very helpful to develop nanoparticle

species, which are taken up specifically by target cells and

exploit their maximum potential.
In this study differently modified silica coated superparamagnetic iron oxide nanoparticles (SPIONs) and silica coated iron
oxide nanoparticles (SCIONs), which were all comparable in
their primary size and surface charge, were tested in HeLa cells
as a model cell line. To elucidate, which molecular pathways
are involved in their endocytosis, well-known endocytotic
mechanisms [26-28] were inhibited by specific knockdown of
key proteins via siRNA (Figure 1).

Experimental
Superparamagnetic iron oxide nanoparticles
(SPIONs)
SPIONs were provided and characterized by MagForce AG.
SPIONs with an iron oxide core of 15 nm and a silica shell of
5 nm were modified by coupling the respective functional
groups as an ethoxy- or rather methoxysilane to the free
hydroxy groups of the surface (Figure 2). These modifications
resulted in different physicochemical properties referring to
SPIONs surface charge and their size distribution under physiological conditions (Table 1). The primary particle size was
determined by transmission electron microscopy (EM906,
Zeiss). The zeta potential and the average hydrodynamic diameter in physiological environment were measured by dynamic
light scattering (Zetasizer, Malvern Instruments Ltd). Due to the

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]).

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Table 1: Surface modification of SPIONs and their physical properties at room temperature in aqueous dispersion (pH 7, DLS = dynamic light scattering).

Surface modification

Linker structure

Zeta potential

Average diameter (DLS)

None (pure silica)

59 mV

136 nm

Carboxylic acid

47 mV

157 nm

Polyethylene glycol

14 mV

133 nm

Medium (DMEM, Invitrogen, Cat. No. 31885023), supplemented with 10% FBS, and cultivated in an incubator at 37 C
and 5% CO2.

Transfection procedure and efficiency

Lipofectamine 2000 transfection reagent

Figure 2: Schematic overview of SPION structure.

synthesis route, often more than one iron core was enclosed
during growth of the silica shells. This caused aggregation and
therefore the nanoparticle suspensions were polydisperse.
The tested modifications were carboxylic acid- and PEGsilanes (Cat. No. SIC2264.0 and SIM6492.7, ABCR GmbH &
Co. KG, Karlsruhe, Germany). SPIONs had been sterilized and
pyrogen tested by MagForce AG. They were stored at 4 C in
aqueous suspension with an iron concentration of 34 mg/mL.

Fluorescent silica coated iron oxide nanoparticles (SCIONs)

SCIONs were provided and characterized by the National Institute of Health (Maryland, USA). They were monodisperse at
pH 7 and had a hydrodynamic diameter of 17 nm with a surface
charge of 50 5 mV. For detection in confocal fluorescence
microscopy, the fluorescent dye Alexa Fluor 555 was
embedded into their shells. Further details of synthesis and
characterization have been described elsewhere [29,30].

Cell culture
HeLa cells (human cervix carcinoma) were provided by the
group of Professor Haucke (Freie Universitt, Berlin,
Germany). They were grown in Dulbecco`s Modified Eagle

Cells were transfected according to the standard protocol of

Life Technologies. To achieve the optimal transfection efficiency, two transfection rounds on day 1 and 3 after cell plating
were performed. In preliminary experiments the knockdown
technique was optimized to cause no cell death by applying
different ranges of transfection reagent with different amounts
of siRNA. The siRNAs were purchased from Eurofins MWG
Operon (Ebersberg, Germany). The sequences are displayed in
Table 2. With the exception of Flotillin-1 all sequences where
created and established by the group of Professor Haucke (Freie
Universitt, Berlin, Germany).

Table 2: Sequences of siRNAs used for transfection with Lipofectamine 2000 transfection reagent.

Oligo name
(siRNA)

Oligo details (sense strand)

Flotillin-1a
Caveolin-1
Clathrin heavy
chain
Dynamin 2
Nonsense control

5-CACACUGACCCUCAAUGUC-3
5-CCUGAUUGAGAUUCAGUGC-3
5-AUCCAAUUCGAAGACCAAUTT-3

aSequence

5-GCAACUGACCAACCACAUC-3
5-GUAACUGUCGGCUCGUGGUTT-3

from Glebov et al. [31].

Dharmacon SMARTpool technology

Cells were transfected according to the protocol Thermo
Scientific DharmaFECT Transfection Reagents - siRNA Transfection Protocol and DharmaFECT 1 siRNA Transfection
Reagent (Thermo Fisher Scientific, Cat. No. T-2001-01) was
used. Information about the siRNA-mix (SMARTpool) used is
shown in Table 3.

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Table 3: siRNA-pools used to transfect HeLa cells (Dharmacon

SMARTpool).

SMARTpool

Catalog number

ON-TARGETplus SMARTpool-Human
PIP5K2
ON-TARGETplus SMARTpool-Human
CDC42

L-006778-00-0005
L-005057-00-0005

Knockdown efficiency
To demonstrate effective knockdown of target proteins, transfected cells were collected in every single experiment. The
expression level of target proteins was determined in comparison to non-transfected control cells by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) of cell lysates
followed by Western Blot and detection of proteins through
specific antibodys. -Actin served as a housekeeping protein to
ensure comparable amounts of proteins in every sample. Primary antibodies used in this study are shown in Table 4.

Nanoparticle exposure
Cells were grown to 7080% confluence. Before every single
experiment, nanoparticles were prewarmed to 37 C and treated
with ultrasound for 10 min to avoid sedimentation and aggregation. SPIONs were diluted with cell culture media to a concentration of 50 g Fe/mL while SCIONs were diluted to 5 g/mL.
The concentration of the SCIONs was chosen after preliminary
experiments. It could be shown that 5 g Fe/mL provides the
lowest background fluorescence combined with a good intracellular signal. In both setups the cell culture media contains
SCIONs in excess to the internalization rate of the cells. Afterward cells were exposed to nanoparticles for 24 h (SPIONs) or
4 h (SCIONs). To ensure natural behavior of nanoparticles in
cell culture media, the plates were left without any movement
or shaking during the exposition. During the incubation time of
cells with nanoparticles no or only minor differences of the cell
numbers were observed, which confirmed no severe impact of
the treatment on cell viability within the observation period.

Quantitative iron analysis

For quantitative determination of iron, which was taken up by
cells and not attached to the plastic surface or to the outer cell
membrane, cells were washed two times with prewarmed cell
culture medium with all additives. Preliminary tests had shown,
that full medium at 37 C removed extracellular adhered
nanoparticles very effective compared to PBS and medium
without additives. To remove the contaminative iron, cells were
rinsed thoroughly with full medium without detaching cells
from the culture surface. Afterward cells were detached with
trypsin/EDTA, counted and cell pellets were resuspended in
concentrated hydrochloric acid. Treatment with hydrochloric
acid and ultrasound for 10 min destroyed the cells and dissociated the iron cores of SPIONs. The iron content of the samples
was then determined by a photometric assay (Spectroquant,
Merck) and by ICPMS (iCAP 6000, Thermo Scientific).
Experiments were repeated three times.

Fluorescence microscopy
Spinning disk confocal microscopy was used to detect SCIONs
inside cells. The applied system was the Zeiss Axiovert 200Mbased spinning disc confocal microscope (PerkinElmer Life
Sciences Inc., MA, USA). Microscopy and quantitative
analyses were performed with the software Volocity (Improvision Inc.). For quantitative determination of SCIONs per cell at
least 140 single cells were analyzed in single layers in each
experiment. To calculate an average fluorescence intensity of
SCIONs for a whole cell population, the overall fluorescence of
the dye Alexa Fluor 555 in every single cell was determined.
Therefore the sensitivity of the nanoparticle channel was
adjusted to the distinct vesicles containing SCIONs. To exclude
background fluorescence of extracellular adherent SCIONs,
single cells were marked by the help of fluorescently labeled
transferrin (Transferrin From Human Serum, Alexa Fluor 488
Conjugate, Invitrogen, Cat.No. T-13342). After that the mean
intensity of the SCION fluorescence per cell was determined.
This procedure was the same for every sample. Experiments
were repeated four times.

Table 4: Primary antibodies with source and dilution used for detection of target proteins.

Antibody

Source

Catalog number

Dilution

Caveolin-1 (N20)
Purified mouse Flotillin-1
Cdc42 polyclonal
Monoclonal anti-PIP5K2A
Monoclonal anti--actin
Anti-Dynamin II polyclonal
Antibody Clathrin

Santa Cruz Biotechnology

BD Transduction
Thermo Scientific
Sigma-Aldrich
Sigma-Aldrich
Thermo Scientific
Haucke group (FU Berlin,
Germany)

sc-894
610820
PA5-17544
WH0005305M1-100UG
A5441-2ML
PA1-661
unknown

1:400
1:100
1:1000
1:350
1:5000
1:1000
1:500

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Statistics

Bare SPIONs with silica shell

To proof significance of detected differences between two

populations, unpaired two-tailed t-tests (confidence interval
= 95%, p value <0.05) were performed by the help of the software GraphPad Prism 5 (GraphPad Software Inc.).

Non-transfected control cells internalized 39.2 1.5 pg Fe/cell

in 24 h, while cells with a knockdown of Caveolin-1 contained
only 28.4 1.0 pg Fe/cell. Hence, knockdown of Caveolin-1
decreased endocytosis of nanoparticles by 27% (Figure 4). This
effect is statistical significant ( = 95%, p = 0.0041). Knockdown of Flotillin-1 (39.2 1.8 pg Fe/cell) and Clathrin
(35.4 0.6 pg Fe/cell) showed no significant difference in the
iron content compared to control cells (Figure 4).

Results
Transfection efficiency
Knockdown of target proteins was confirmed by determination
of the expression level of the respective proteins in transfected
and non-transfected cells in every single experiment.
Semi-quantitative determination of proteins in cell lysates
showed efficient knockdown for Clathrin, Dynamin 2,
Flotillin-1, PIP5K and CDC42 (Figure 3). In case of
Caveolin-1 around 5060% knockdown efficiency has been
achieved (Figure 3a). This was sufficient to detect an effect on
the endocytosis of nanoparticles. -Actin was used as a housekeeping protein to ensure comparable amounts of proteins in
every sample.

Knockdown of Caveolin-1, Flotillin-1 and

Clathrin
Endocytosis through Caveolin-1, Flotillin-1 and Clathrin was
inhibited by knockdown of the respective protein by specific
siRNAs via the Lipofectamine technology. HeLa cells were
incubated for 24 h with SPIONs (50 g Fe/mL) which were
either caboxylated, PEGylated or which had no further modifications on their silica shell. Uptake of nanoparticles was
measured by dissolution of cell pellets and nanoparticles in
concentrated hydrochloric acid, followed by quantitative
determination of the iron content by a photometric assay and
ICP.

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 g/mL, error
bars: SEM, n = 3).

Carboxylated SPIONs
HeLa cells with a knockdown of Caveolin-1 contained 23% less
carboxylated SPIONs compared to non-transfected control cells

Figure 3: Representative X-ray films with knockdown efficiency of target proteins (red labels), -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 PIP5K.

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(Figure 5). The iron levels per cell amounted to 12.0 0.5 pg
for Caveolin-1 depleted cells and 15.5 1.2 pg for control cells.
This effect is statistically significant ( = 95%, p = 0.0466).
Knockdown of Flotillin-1 (17.9 1.9 pg Fe/cell) and Clathrin
(17.7 1.9 pg Fe/cell) resulted in slightly more nanoparticles
inside the cells. However, this effect is statistically not significant (Figure 5).

an increase of 45% compared to control cells (Figure 6). All

these effects are not statistically significant.

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 g/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 g/mL, error
bars: SEM, n = 3).

PEGylated SPIONs
Compared to control cells (17.7 2.5 pg Fe/cell), HeLa cells
contained 33% less iron (11.8 0.8 pg Fe/cell) if the
expression level of Caveolin-1 was reduced (Figure 6). Knockdown of Flotillin induced no detectable difference
(19.4 2.1 pg Fe/cell), while knockdown of Clathrin produced
an elevated iron content per cell of 25.6 1.7 pg and therefore

SCIONs in confocal fluorescence microscopy

SCIONs were comparable to SPIONs in their primary size and
surface charge. Because of the fluorescent dye Alexa Fluor
555, which was embedded into their cells, these nanoparticles
were intracellular detectable with confocal fluorescence
microscopy. To avoid heavy background fluorescence of extracellular adherent SCIONs, the iron concentration used was
lowered to 5 g/mL and the incubation time was shortened to
4 h. After incubation with nanoparticles, cells were imaged (at
least 140 single cells per single experiment) and background
fluorescence was eliminated (Figure 7). Finally, the average
sum-fluorescence-intensity of SCIONs per cell was calculated.

Figure 7: Fluorescence image of Hela cells which were incubated with SCIONs (iron concentration 5 g/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.

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6.8 10 6 0.8 10 6 average sum-intensity per cell was

detected in HeLa cells, which were treated with siRNA against
Caveolin-1 (Figure 8). Control cells showed 1.2 107 0.1
107 sum-intensity per cell and therefore 41% more than cells
with a knockdown of Caveolin-1. This effect was statistically
significant ( = 95%, p = 0.0019). Depletion of Dynamin 2,
Flotillin-1 and Clathrin showed no effect on the detectable fluorescence of SCIONs inside the cells (Figure 8). Although the
total amount was slightly increased for all three knockdowns,
this effect is statistically irrelevant and can be ignored.

Figure 9: Iron content of control and transfected HeLa cells in pg/cell

after 24 h incubation with PEGylated SPIONs; target proteins: CDC42,
PIP5K (iron concentration 50 g/mL, error bars: SEM, n = 3).

Summary

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 g/mL, n = 4).

Endocytosis mediated by CDC42 and PIP5K

Knockdown of CDC42 and PIP5K was realized through transfection with specific siRNA compositions via the Dharmacon
SMARTpool technology. As described above, cells were incubated with PEGylated SPIONs for 24 h and iron content per cell
was determined. To distinguish between nanoparticles inside
the cells and nanoparticles, which are attached to the outer cell
membrane, control experiments at 4 C were performed.
0.8 0.1 pg Fe/cell were detected and subtracted from every
measurement. Carboxylated SPIONs were not included because
of their very similar behavior compared to the PEGylated
SPIONs.
Control cells internalized 34.8 0.4 pg Fe/cell (Figure 9).
Depletion of CDC42 decreased this level to 18.6 1.6 pg Fe/
cell. Hence, the difference between control cells and cells
without CDC42 amounts to 46%. This effect is statistically
highly significant ( = 95%, p = 0.0006). Knockdown of
PIP5K resulted in 29.4 4.2 pg Fe/cell, which is 15% less
compared to control cells. But due to the high standard deviation, this effect is not statistically significant ( = 95%,
p = 0.2773).

Knockdown of Caveolin-1 decreased the ability of HeLa cells

to internalize nanoparticles. Depending on the surface modification of SPIONs or SCIONs and the experimental design, the
endocytosis of nanoparticles dropped between 23% and 41%
compared to non-transfected control cells. Depletion of CDC42
resulted in a reduced endocytosis of PEGylated SPIONs by
46% compared to control cells. Knockdown of other target
proteins like Dynamin 2, Clathrin, Flotillin-1 and PIP5K did
not show significant effects on the internalization behavior of
HeLa cells in vitro.

Discussion
The aim of the study was to elucidate, how human cancer cells
internalize iron oxide nanoparticles with silica shells, which
have no target function for a special application or receptor.
Therefore the human cervical cancer cell line HeLa was chosen
as a model cell line. Hela cells are a well-established malignant
cell line, which was widely used to study the uptake of iron
oxide nanoparticles [18,21,24,32], gold nanoparticles [33,34]
and other particle systems like quantum dots [35] or polymer
particles [36,37]. To gain insights into the molecular mechanisms, which are involved in the endocytosis of iron oxide
nanoparticles, and how the uptake is influenced by parameters
like size and surface composition of nanoparticles would be
very useful in the development of therapeutic approaches.

Involvement of Caveolin-1, Flotillin-1 and

Clathrin in the endocytosis of SPIONs
The results show, that knockdown of Caveolin-1 reduced endocytosis of unmodified, carboxylated and PEGylated SPIONs by
HeLa cells between 23 and 33% compared to control cells

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(Figure 4, Figure 5 and Figure 6). Therefore this effect was

reproducible when SPIONs with comparable properties in size
and surface charge were used. The effect may be even more
distinct under complete knockdown conditions of Caveolin-1.
Although comparison of different particle species is very difficult due to their varying properties, the involvement of
Caveolin-1 in the endocytosis of nanoparticles by HeLa cells is
consistent with the literature. It was shown, that polyethyleneimine gold nanoparticles around 40 nm [33], gold nanoparticles of 4.5 nm [34] and conjugated polymer nanoparticles [36]
are internalized through Caveolin dependent pathways. The
same was observed for human alveolar epithelial cells and polystyrene nanoparticles around 100 nm [38] as well as polymer
coated gold nanoparticles with a core size around 13 nm [39].
On the other hand there are studies showing the uptake of
different nanoparticles by HeLa cells such as quantum dots
[35], PEG-PLA particles [37] and mesoporous silica particles
[40] exclusively through Clathrin mediated endocytosis and/or
macropinocytosis. This discrepancy can be explained by the
different physicochemical properties of the particles used. Especially in the field of iron-oxide nanoparticles more studies
concerning the endocytotic pathways have to be done to clarify
the underlying principles.
Significant influences of Flotillin-1 and Clathrin were not
detectable. However, when the cells with knockdown of
Flotillin-1 and Clathrin were incubated with SPIONs for 24 h,
their intracellular iron content was slightly increased compared
to the control cells (Figure 5, Figure 6). This points to a possible
compensatory upregulation of other endocytotic mechanisms, as
it was shown for HeLa cells [41] as well as for MDCK and
HeLa cells incubated with PEG-PLA nanoparticles [37,42].

Involvement of Caveolin-1, Flotillin-1, Clathrin

and Dynamin 2 in the endocytosis of SCIONs
To verify the results gained with SPIONs, SCIONs with comparable properties regarding their chemical composition, size and
surface charge were tested with confocal fluorescence
microscopy. Dynamin 2 was included as a new target protein
because Dynamin 2 has been identified to act together with
Caveolin-1 and in other routes of endocytosis [27,43].
The results show, that knockdown of Caveolin-1 decreased
endocytosis of SCIONs by HeLa cells by 41% within 4 h incubation time, while knockdown of Flotillin-1 and Clathrin
showed no significant effects (Figure 8). This confirms the
previous findings regarding the endocytosis of SPIONs. The
effect of Caveolin-1 in the endocytosis of SCIONs is higher
compared to SPIONs. On the other hand this is possibly due to
the different experimental setups, on the other hand SCIONs
provided a narrow size distribution, which could support intra-

cellular uptake through distinctive pathways. Potentially the

heterogeneous size distribution of SPIONs is also the reason for
the relatively low effect of Caveolin-1 compared to other findings in HeLa cells [33,34].
Interestingly, knockdown of Dynamin 2 showed no effect on
the endocytosis of SCIONs by HeLa cells. This could be an indication for an unknown, alternative uptake mechanism, which
is dependent on Caveolin-1 but independent from Dynamin 2.
Because it is known, that Dynamin 2 plays an important role in
the constriction of caveolae-coated vesicles from the inner cell
membrane [27,43], another possible explanation is, that
SCIONs accumulate in caveolae-coated vesicles at the cell
membrane without their detachment when Dynamin 2 is
depleted. These SCIONs would not have been removed before
quantitative analysis. Further experiments have to be conducted
to test these hypotheses.

Involvement of CDC42 and PIP5K in the

endocytosis of SPIONs
The observed effect of Caveolin-1 on the endocytosis of
SPIONS and SCIONs by HeLa cells did not fully explain, how
these particles are taken up. So other candidates of the endocytotic machinery had to be tested. Knockdown of CDC42
reduced endocytosis of PEGylated SPIONs by 46% (Figure 9).
This effect was highly significant and visible by eye before
quantitative iron analysis because of the light-colored cell
pellet. The important role of CDC42 is also interesting in the
context of the observed effect of Caveolin-1, because it was
shown, that depletion of Caveolin-1 in the epithelial ovarian
hamster cell line CHO-K1 enhances fluid phase endocytosis
dependent on CDC42 [44]. This indicates a possible compensation of the Caveolin-1 knockdown in the experiments with
SPIONs and SCIONs. CDC42 is not only related to Flotillin-1,
it is involved in many other cellular processes including
macropinocytosis [45], therefore explaining its relevance in the
uptake of polydisperse SPIONs. Depletion of PIP5K caused no
significant effect.

Conclusion
This study shows for the first time, that Caveolin-1 and CDC42
play an important role in the endocytosis of SCIONs and
SPIONs with negative surface charge and a primary diameter
around 17 to 30 nm in HeLa cells in vitro. Depending on the
nanoparticle used, 69 to 87% in addition of the endocytosed
particles were taken up through Caveolin-1 and CDC42 dependent pathways. Because of the heterogeneous nanoparticle
suspensions, involvement of more than one specific pathway is
not surprising. Endocytosis through Caveolin-1 and CDC42 is
characterized by vesicles of 30 to 80 nm [27,28], which
excludes bigger agglomerates from uptake. For future experi-

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Acknowledgements
The authors would like to thank Dr. Fischler and Dr. Bumb for
the preparation of nanoparticles. This work was funded by the
Deutsche Forschungsgemeinschaft, priority program SPP1313.

doi:10.1016/S0142-9612(02)00440-4
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