Supplementary Information
Experimental set-up. SPIO nanoparticles were fluorescently labeled with Alexa Fluor488
hydrazide according to the manufacturer’s instruction (Invitrogen). Alexa Fluor488 hydrazide
is an amine-reactive fluorophore which is covalently attached the label to the carboxy groups
on SPIO. Once formed, the covalent bond between the label and the nanoparticle is extremely
stable. Physicochemical parameters of the particles have previously been described.1
Carboxydextran-coated superparamagnetic iron oxide nanoparticles (SPIO) have mean size of
an iron oxide core of about 5 nm and mean hydrodynamic diameters of about 60 nm. PC-3
cells were grown in IBIDI µ-slides (Munich, Germany) at concentrations of 3 × 104
cells/channel in 120 µl growth medium (F12K supplemented with 10 % FCS, 50 U penicillin
and 500 µg/ml streptomycin) then incubated with SPIO (100 µg Fe/ml) either in the presence
or absence of the permanent cubic magnet (NdFeB, 5 mm edge length, magnetic field strength
of 1210 mT). Cell membranes were stained with CellMask™ Deep Red (red dye, Invitrogen).
Fluorescence images were recorded with the acquisition software ZEN 2009 using a Zeiss
LSM710 confocal scanning microscope with a 63× 1.4NA immersion oil objective. ImageJ
software (NIH) was used for image processing and movie reconstruction.
Studies of NP uptake and measurement of intracellular iron. To determine the mechanism
of
NP uptake, PC-3 cells (1 x 106 cells/ml) were pre-incubated with different pathway
inhibitors (macropinocytosis, 2 µM rottlerin for 30 min2; pinocytosis, 100 µg/ml colchicine
for 2 h3; phagocytosis, 10 µg/ml cytochalasin B for 2 h3; clathrin-mediated endocytosis, 200
µM monodansylcadaverine for 10 min4; caveolae-mediated endocytosis, 50 µg/ml nystatin for
15 min5; dynamin-dependent endocytosis, 80 μMdynasore for 30 min6) and treated with 100
µg/ml of particles for 6 h. Nanoparticle uptake was analyzed by spectroscopic measurement
1
of intracellular iron. Briefly, after stimulation, the cells were washed, dehydrated at 110°C
overnight and iron was dissolved in 100 mM citric acid. Fe3+ was reduced to Fe2+ with
ascorbic acid, treated with bathophenanthroline disulfonic acid, and iron bathophenanthroline
disulfate was analyzed spectroscopically7 using FeCl2 for the generation of standard curves.
Raster image correlation spectroscopy (RICS) analysis for the determination of
nanoparticle velocities. Single-point fluorescence correlation spectroscopy (FCS) allows
measurements of fast diffusion and dynamic processes in the microsecond-to-millisecond
time range. Raster image correlation spectroscopy (RICS), rapidly measures during the scan
many focal points within the cell providing the same concentration and dynamic information
of FCS as well as information on the spatial correlation between points along the scanning
path. Basic principles of RICS have previously been described8 and we used a published
approach for RICS quantification.9 A Zeiss LSM 710 laser scanning microscope equipped
with an incubation chamber and a 63×1.4NA oil immersion lens was used. Data acquisition
parameters were optimized using control measurements of Rhodamine R110. The 488-nm
laser was used with 0.5 % of transmission power and the signal was detected using a longpass emission filter (505 nm). The pinhole was set to 1 airy unit, detector gain was set to 700
V, and offset was optimized to 0. Images were taken at 512 × 512 and 16 bits. The excitation
volume was measured by taking a z-stack of a subresolution of a 175-nm diameter fluorescent
bead and fitting with a Gaussian in Mathcad software. Pixel size was 55 nm to ensure
sufficient oversampling within the size of the excitation volume.
Figure S1. Experimental setup. A cubical magnet with the length a=5mm was placed
below the cell culture.
2
Fluorescence images were taken with the acquisition software ZEN 2009 using a Zeiss
LSM710 confocal scanning microscope with a 63× 1.4NA immersion oil objective. ImageJ
software (NIH) was used for image processing and movie reconstruction.
Figure S2. Uptake of SPIO in the presence of different inhibitors.
PC-3 cells were pretreated with different uptake inhibitors, and subsequently incubated with
SPIO (100 µg/ml) for 6 h. Iron was measured by spectrometric detection after conversion to
iron bathophenanthroline disulfate in the presence of ascorbic and bathophenanthroline
disulfonic acid. Results are presented as mean ± SEM, n = 3, *p< 0.05, **p< 0.01, NewmanKeuls test. Pathway inhibitors: macropinocytosis, 2 µM rottlerin for 30 min; pinocytosis, 100
µg/ml colchicine for 2 h; phagocytosis, 10 µg/ml cytochalasin B for 2 h; clathrin-mediated
endocytosis, 200 µM monodansylcadaverine for 10 min; caveolae-mediated endocytosis, 50
µg/ml nystatin for 15 min; dynamin inhibitor, 80 µM dynasore for 30 min.
To evaluate the mechanistic nature of the NP uptake, PC-3 cells were pretreated with different
uptake inhibitors prior to incubation with the particles. No significant changes in nanoparticle
uptake were observed after pretreatment with inhibitors of phagocytosis, pinocytosis,
macropinocytosis or caveolin-mediated endocytosis (Fig. S2). The most profound inhibition
of accumulation of SPIO occurred in the presence of inhibitors of dynamin- and clathrindependent endocytosis, dynasore and monodansylcadaverine, suggesting that dynamin- and
clathrin-dependent endocytosis may play a major role in the uptake of SPIO particles.
Figure S3. Kinetics of SPIO endocytosis by PC-3 cells with or without a magnetic field.
The images show uptake of fluorescently labeled SPIO by PC-3 cells using Zeiss LSM710
confocal scanning microscope. PC-3 cells were treated with Alexa488-SPIO (100 µg Fe/ml,
3
green). Cell membranes were labeled with CellMask™ Deep Red (red). ImageJ (NIH)
software was used for image processing. Original magnification 400x, scale bar 40 µm.
Movie S1. The movie shows uptake of fluorescently labeled SPIO by PC-3 cells in the
presence of a permanent cubic magnet (NdFeB, a cube with 5 mm edge length and a magnetic
field strength of 1.21T) in the time frame from 1-128 min. Imaging was done using a Zeiss
LSM710 confocal scanning microscope. PC-3 cells were treated with Alexa488-SPIO (100 µg
Fe/ml, green). Cell membranes were labeled with CellMask™ Deep Red (red). ImageJ (NIH)
software was used for image processing. Original magnification 400x, scale bar 40 µm.
Movie S2.The movie shows uptake of fluorescently labeled SPIO by PC-3 cells in the
presence of a permanent cubic magnet (NdFeB, a cube with 5 mm edge length and magnetic
field strength of 1.21 T) in the time frame from 130-150 min. Imaging was done using a Zeiss
LSM710 confocal scanning microscope. PC-3 cells were treated with Alexa488-SPIO (100 µg
Fe/ml, green). Cell membranes were labeled with CellMask™ Deep Red (red). ImageJ
software was used for image processing. Original magnification 630x, scale bar 8 µm.
Movie S3. The movie shows uptake of fluorescently labeled SPIO by PC-3 cells in the
absence of the permanent magnet in the time frame from 1-128 min. Imaging was done using
a Zeiss LSM710 confocal scanning microscope. PC-3 cells were treated with Alexa488-SPIO
(100 µg Fe/ml, green). Cell membranes were labeled with CellMask™ Deep Red (red).
ImageJ software was used for image processing. Original magnification 400x, scale bar 40
µm.
Movie S4. The movie shows uptake of fluorescently labeled SPIO by PC-3 cells in the
absence of the permanent magnet in the time frame form 130-150 min. Imaging was done
using a Zeiss LSM710 confocal scanning microscope. PC-3 cells were treated with Alexa4884
SPIO (100 µg Fe/ml, green). Cell membranes were labeled with CellMask™ Deep Red (red).
ImageJ software was used for image processing. Original magnification 630x, scale bar 8 µm.
Movie S5. The uptake of fluorescently labeled SPIO particles by PC-3 cells in the absence of
magnet (3D reconstruction). PC-3 were treated with SPIO (100 µg/ml) labeled with Alexa
Fluor 488 (green) for 6 h. Fluorescence images were recorded with the acquisition software
ZEN 2009 using a Zeiss LSM710 confocal scanning microscope with a 63× 1.4NA
immersion oil objective. ImageJ software (NIH) was used for image processing and movie
reconstruction. Original magnification 630x. The membrane was stained with CellMask™
Deep Red (red dye).
Movie S6. The uptake of fluorescently labeled SPIO particles by PC-3 cells in the presence of
magnet (3D reconstruction). PC-3 were treated with SPIO (100 µg/ml) labeled with Alexa
Fluor 488 (green) for 6 h. Fluorescence images were recorded with the acquisition software
ZEN 2009 using a Zeiss LSM710 confocal scanning microscope with a 63× 1.4NA
immersion oil objective. ImageJ software (NIH) was used for image processing and movie
reconstruction. Original magnification 630x. The membrane was stained with CellMask™
Deep Red (red dye).
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