Endocytosed nanoparticles hold endosomes and stimulate binucleated cells formation
Xia et al. Particle and Fibre Toxicology
Endocytosed nanoparticles hold endosomes and stimulate binucleated cells formation
Lin Xia 0 2
Weihong Gu 0 2
Mingyi Zhang 0 2
Ya-Nan Chang 0 2
Kui Chen 0 2
Xue Bai 0 2
Lai Yu 0 2
Juan Li 0 2
Shan Li 0 1 2
Gengmei Xing 0 2
0 CAS Key Laboratory for Biomedical Effects of Nanomaterial & Nanosafety, Institute of High Energy Physics, Chinese Academy of Science (CAS) , Beijing 100049 , China
1 School of Bioscience & Bioengineering, South China University of Technology (SCUT) , Guangzhou 510006 , China
2 Authors' Information Lin Xia (first author): Candidate of PhD, major in bioinorganic chemistry, Institute of High Energy Physics, Chinese Academy of Sciences; Weihong Gu(co-author): Candidate of PhD, major in bioinorganic chemistry, Institute of High Energy Physics, Chinese Academy of Sciences; Mingyi Zhang(co-author): PhD, major in bioinorganic chemistry, Institute of High Energy Physics, Chinese Academy of Sciences; Yanan Chang(co-author): PhD,Research Assistant, Institute of High Energy Physics, Chinese Academy of Sciences; Kui Chen(co-author): MD, Laboratory Assistant, Institute of High Energy Physics, Chinese Academy of Sciences; Xue Bai(co-author): Candidate of PhD, major in bioinorganic chemistry, Institute of High Energy Physics, Chinese Academy of Sciences; Lai Yu(co-author): Candidate of MD, major in clinical medicine, Jinzhou Medical University, Guest Researcher in Institute of High Energy Physics, Chinese Academy of Sciences; Juan Li(co-author): PhD, Associate Researcher, Institute of High Energy Physics, Chinese Academy of Sciences; Shan Li(co-author): PhD, Associate Professor, South China University of Technology, Guest Researcher in Institute of High Energy of Physics, Chinese Academy of Sciences; Gengmei Xing(
Background: Nanotechnology developed rapidly in cellular diagnosis and treatment, the endocytic system was an important pathway for targeting cell. In the research of developing macrophages as drug carriers or important therapeutic targets, an interesting phenomenon, internalized nanoparticles induced to form binucleated macrophages, was found although the particles dose did not cause obvious cytotoxicity. Results: Under 25 μg/ml, internalized 30 nm polystyrene beads(30 nm Ps nanoparticles) induced the formation of binucleated macrophages when they entered into endosomes via the endocytic pathway. These internalized 30 nm Ps nanoparticles (25 μg/ml) and 30 nm Au-NPs (1.575 ng/ml) also induced markedly rise of binucleated cell rates in A549, HePG-2 and HCT116. This endosome, aggregated anionic polystyrene particles were dispersed and bound on inner membrane, was induced to form a large vesicle-like structure (LVLS). This phenomenon blocked transport of the particles from the endosome to lysosome and therefore restricted endosomal membrane trafficking through the transport vesicles. Early endosome antigen-1 and Ras-related protein-11 expressions were upregulated; however, the localized distributions of these pivotal proteins were altered. We hypothesized that these LVLS were held by the internalized and dispersed particles decreasing the amount of cell membrane available to support the completion of cytokinesis. In addition, altered distributions of pivotal proteins prevented transfer vesicles from fusion and hampered the separation of daughter cells. Conclusions: 30 nm Ps nanoparticles induced formation of LVLS, blocked the vesicle transport in endocytic system and the distributions of regular proteins required in cytokinesis which led to binucleated cells of macrophages. Markedly raised binucleated rate was also observed in human lung adenocarcinoma epithelial cell line(A549), human hepatoma cell line(HePG-2) and human colorectal cancer cell line(HCT116) treated by 30 nm Ps nanoparticles and Au-NPs.
Nanoparticles; Endocytic; Endosome; Large vesicle-like structures; Binucleated cell
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Background
Nanotechnology developments are facilitating the use of
engineered particles to diagnose and treat diseases on
the cellular level. Many researchers have investigated
various nanoparticle properties including surface charge,
size, shape, and rigidity, while others have studied how
to control the interaction of particles with the cell
membrane to allow endocytic transport into target cells
[1–3]. While many reports have confirmed the
biocompatibility of nanoparticles, one major question is whether
they will interfere in the native physiological functions
of the endocytic system, such as membrane trafficking.
The eukaryotic endocytic system consists of
pleiotropic intracellular organelles including endosomes.
Recent studies have revealed that the endosome, which was
a membrane pool, participated in membrane trafficking
to maintain membrane cellular equilibrium. Disruption
of this balance would affect cell physiologic functions
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and even result in cytokinesis failure [4]. Endosomes are
pivotal organelles in the endocytic pathway, they
transport cargoes to the lysosome and serve as the primary
site for the returning and transport of plasmalemma and
protein sorting [5]. Previous studies have shown that
cargo proteins mediated the engulfment vesicles passing
through the endosomes [6]. Nanoparticles enter cells by
different endocytic pathways and are transported into
endosomes via endosomal-lysosomal pathway. It remains
unclear whether nanoparticles with certain properties
could impact the physiological functions of endosomes.
A growing number of researchers have shown interest
in developing macrophages as drug carriers or important
therapeutic targets with increasing understandings of
macrophage’s biological roles in disease. Endocytosis is a
fundamental function of macrophages that facilitate the
destruction and subsequent degradation of ingested
microbes or allogenic materials. To perform this function,
macrophages must rapidly replenish their cell surface
membrane from intracellular membrane pools
constructed with recycling endosomes. Fielding and Wilson
groups have demonstrated that endosomes are also
involved in mediating cellular cytokinesis and have also
shown that sufficient membrane and pivotal proteins have
to be available to allow the division of a mother cell into
two daughter cells. We found that when internalized
30 nm Ps nanoparticles entered endosomes in
macrophages, it restricted the recycling of endosomes back to
the cell surface and binucleated cells become visible under
the microscope. Although the particles aren’t necessarily
toxic, these binucleated cells are genetically unstable.
Therefore, we investigated how nanoparticles induced
formation of binucleated cells following their endocytosis.
Methods
Cell culture
The murine macrophage cell line RAW 264.7, human
cancer cell lines A549, HePG-2 and HCT116 were
purchased from the Cancer Hospital at the Chinese Academy
of Medical Sciences (Beijing, China). The murine
macrophage cell line RAW264.7 was cultured in Dulbecco’s
minimal essential media (DMEM)/F-12 (Hyclone
Laboratories, Logan, UT) supplemented with 10% fetal bovine
serum (FBS) in a humidified atmosphere of 5% CO2 at
37 °C. Human cancer cell lines A549, HePG-2, HCT116
cell lines were cultured in Dulbecco’s minimal essential
media (DMEM)/HIGH GlUCOSE (Hyclone Laboratories,
Logan, UT) supplemented with 10% fetal bovine serum
(FBS) in a humidified atmosphere of 5% CO2 at 37 °C.
Nanoparticle preparation
30 nm Ps nanoparticles labelled with FITC were
purchased from Sigma (St. Louis, MO) and subsequently
characterized using a scanning electron microscope
(SEM; model S-4800; Hitachi, Tokyo, Japan). The
NicompTM 380 ZLS particle size/zeta potential analyzer
(Agilent Technologies, Santa Clara, CA) was used to
determine the zeta potential of the particles with or
without sonication. The measurements were performed in
complete cell culture media at pH 7.4. Synthesis of
30 nm Au nanoparticles (Au-NPs) was according to
citrate reduction method [7] and subsequently
characterizing of the nanoparticles used SEM and DLS.
Measure of cell viability
200 ul RAW 264.7 cells suspension (1 × 104 cells/ml)
were placed in every well of 96-well culture plate and
cultured for 24 h, then the medium was respectively
replaced with fresh medium containing 5, 25, and 50 μg/
ml 30 nm Ps nanoparticles with 10-well replicates. After
24 or 48 h, cell viability was measured using Cell
Counting Kit (CCK-8) ((Dojindo Laboratories, Japan)
according to the manufacturer’s instruction. Following,
viabilities of A549, HePG-2 and HCT116 cells treated by
30 nm Ps nanoparticles (25 μg/ml) and 30 nm Au-NPs
(0.7875,1.575,3.15 ng/ml) was respectively measured.
Cellular uptake of particles
A 1-ml RAW264.7 cells suspension (1 × 105 cells/ml)
was added to a confocal petri dish and cultured for 24 h,
then the medium was replaced with fresh medium
containing 25 μg/ml 30 nm Ps nanoparticles. Cells were
cultured at 37 °C in a humidified atmosphere of 5% CO2
for 1 h, then were washed with PBS and fixed with 4%
paraformaldehyde for 30 min at room temperature.
Treated cells were observed and imaged with a confocal
laser scanning microscope (Perkin-Elmer spinning disc
confocal microscope [Waltham, MA] with a Nikon
microscope [Nikon, Tokyo, Japan]) at a magnification of
400×. In the second group, the cells were cultured in
particle-containing medium at 37 °C for 24 h. A movie
of cells treated by particles for 11 h was taken in the
confocal laser scanning microscope over a 60-min period
(1 frame/min) at a magnification of 400 ×.
Flow cytometer assay and cell counting
RAW 264.7 cells were cultured in medium with 25 μg/
ml 30 nm Ps nanoparticles for 24 h and then were
divided into two groups. Fist group, cells were digested in
2 ml trypsin. After 2 min, 2 ml fresh DMEM/F-12
supplement with 10% fetal bovine serum (FBS) was added
to end the treatment, and the medium containing the
treated cells was centrifuged (1200 rpm, 3 min). The
cells were collected and washed with PBS (0.01 M,
pH 7.2–7.4) three times, and then cold alcohol was
added for 1–2 h to fix the cells. Fixed cells were
collected through centrifugation (1200 rpm, 3 min), and
propidium iodide was added to the tubes for 30 min to
dye the DNA. The DNA quantities of treated cells were
detected with a flow cytometer (Accuri C6 BD, Ann
Arbor, MI) in the FL-2H channel. Second group, cells
were washed with PBS and fixed with 4%
paraformaldehyde for 30 min at room temperature. Treated and
control cells were observed with a confocal microscope
[Nikon Ti-E imaging system, Tokyo, Japan] and the
binucleated macrophages were counted in ten stochastic
views. The rate of binucleated cells was calculated and
the cell was imaged at a magnification of 400 ×.
A549, HePG-2 and HCT116 cells were respectively
cultured in medium with 25 μg/ml Ps nanoparticles for
48 h(data not shown), and then cells were washed with
PBS and fixed with 4% paraformaldehyde for 30 min at
room temperature. Treated and control cells were
respectively observed with a confocal microscope and the
binucleated cells were counted in ten stochastic views.
The number of counted cells was more than 300 for
each kind of cell line. The rate of binucleated cells was
calculated. Image of cell was at a magnification of 400×.
In other treatment, these cells were respectively cultured
in medium with 1.575 ng/ml Au-NPs for 24 h, and then
followed above.
Immunofluorescent imaging
RAW 264.7 cells were cultured in confocal petri dishes
for 24 h, then the medium was replaced with fresh
medium containing 25 μg/ml 30 nm Ps nanoparticles
and the cells were cultured at 37 °C in a humidified
atmosphere of 5% CO2 for 10, 30 and 50 min. Treated
cells were washed with 0.01 M phosphate-buffered saline
(PBS) and fixed with 4% paraformaldehyde for 30 min at
room temperature. Then paraformaldehyde was
aspirated, and the cells were rinsed in PBS three times.
Primary antibodies against early endosomal antigen 1
(EEA1) (CST, Danvers, MA), Ras-related protein-7
(Rab7) (Abcam, Cambridge, UK), lysosomal-associated
membrane protein 1 (LAMP-1) (Abcam, Cambridge,
UK), and Ras-related protein-11 (Rab11) (Abcam,
Cambridge, UK) were used to label EEA1, Rab7,
LAMP1, and Rab11 proteins respectively. Specifically, Rab11
and EEA1 were used to label the cell of division in the
late stages. For these experiments, cells were cultured in
particle-containing medium for 1 h, were then
transferred into fresh medium for 12 h. The dilutions and
incubation times were according to the antibody manuals
provided by the manufacturers. After the primary
antibody incubation, cells were incubated with a secondary
antibody with rhodamine (Abcam, Cambridge, UK) to
identify cellular distributions of EEA1-, Rab7-,
LAMP1-, and Rab11-positive vesicles. Hoechst 33342
(Thermal fisher scientific, Waltham, MA,) was used to
label the nuclei. Prepared petri dishes were observed
and imaged with a confocal fluorescent microscope
(Nikon Ti-E imaging system, Tokyo, Japan) at a
magnification of 400×. Redistributions of the particles
(green), EEA1(red), Rab7(red) and LAMP-1(red) at
10, 30 and 50 min in the cells were probed by
imaging of confocal fluorescence microscope.
Transferrin imaging
RAW 264.7 cells adherent to round coverslips were
preincubated with 25 μg/ml 30 nm Ps nanoparticles in
DMEM/F-12 for 12 h at 37 °C. The medium was
aspirated and discarded, the cells were washed three times
with PBS followed by incubation on ice for 20 min in
DMEM/F-12 medium containing 20 mM glucose and
1% bovine serum albumin (BSA). Next, we added
transferrin-Alexa Fluor 555 (Thermo Fisher Scientific,
Waltham, MA) to the medium at a final concentration
of 2.5% (volume/volume) and incubated at 37 °C for
20 min. Afterward, cells were washed with fresh medium
three times. Transferrin imaging was performed with a
confocal microscope at a magnification of 400 ×.
Western blot of EEA1 and Rab11
After 12 h treatment with 25 μg/ml 30 nm Ps
nanoparticles, RAW 264.7 cells were suspended in PBS by scraping.
Protein samples from whole cells were prepared as
previously described [8]. Bicinchoninic acid (BCA) protein
assay (Beyotime Institute of Biotechnology, Jiangsu,
China) was used to determine protein concentrations in
samples. Each lane in a 10% polyacrylamide SDS gel was
loaded into a 20-μg protein sample, which was then
separated by electrophoresis (100 V). The separated proteins
were transferred from gels to nitrocellulose transfer
membrane (0.2 μm; Whatman, Maidstone, UK) by
electroblotting. The membranes were incubated in 5% non-fat milk
in TBST (Tris-buffered saline and Tween 20) to block
nonspecific binding. The blocked membranes were
incubated with a primary antibody against EEA1 (CST,
Danvers, MA) or Rab11 (Abcam, Cambridge, UK) (following
the manufacturers’ instructions) overnight at 4 °C, then
were rinsed three times for 10 min each in TBST. The
membrane was then incubated with a corresponding
secondary antibody (Santa Cruz Biotechnology, Santa Cruz,
CA) for 40 min at room temperature and washed three
times for 10 min each in TBST. SuperSignal West Pico
(Pierce Biotechnology, Rockford, IL) was used to visualize
the protein bands, which were normalized to control
bands visualized with a monoclonal anti-ß-GAPDH-IgG
antibody (Santa Cruz Biotechnology).
Statistical analysis
Data were analyzed with SPSS ver. 19.0 software (SPSS,
Chicago, IL) using one-way analysis of variance (ANOVA).
Results were validated by performing at least three
independent experiments. The results were expressed as mean
value ± standard error of the mean (SEM). Differences
were considered significant at P < 0.05.
Results
Nanoparticles were internalized by cells
Images of SEM showed that ultra-sonication increased the
number of monodisperse particles (Fig. 1a). DLS assay
showed that Zeta potential of the particles varied from
−14 mV to −50 mV via sonication of 30 min (Fig. 1b).
Characterized Au-NPs with 30 nm diameter was
confirmed by SEM and DLS (Additional file 1: Figure S1, A,
B). After sonication, the Ps nanoparticles labelled with
FITC, was co-cultured with macrophages(RAW264.7 cell
line) for 1 h at 4 °C or 37 °C, respectively. Confocal
fluorescence microscope showed that the presence of green
fluorescence in the cytoplasm of cells cultured at 37 °C
(Fig. 1c), but not in those incubated at 4 °C (data not
shown). Most of the particles located in vesicles after 1 h
(Fig. 1c, a magnified cell was circled by a red line). Large
vesicle-like structures (LVLS, white arrow) with
fluorescence labels were also present in the cytoplasm (Fig. 1d).
Similar phenomenon also presented in this treated cells of
A549, HePG-2 and HCT116 (Fig. 3b, c, d).
Nanoparticles induced the formation of binucleated
macrophages
Under different concentrations of 30 nm Ps nanoparticles,
varied viability of RAW264.7 cell line was detected using
CCK-8 Kit. The varied viability of cells with treated time
extension (24 h to 48 h) didn’t show statistically significant
compared to the control under different concentrations,
but 50ug/ml and the co-culturing time of 48 h depressed
the viability of cells clearly. The rate of the binucleated
macrophages was the highest under the concentration of
25 μg/ml for 24 h (Additional file 1: Figure S2, A, B).
Whereafter, formation of binucleated macrophages was
observed by confocal laser scanning microscope after
12 h cultured in 25 μg/ml nanoparticle-containing
medium. LVLS outlined with green fluorescence
persisted in the cytoplasm, even when two nuclei were
visible in the cell (Fig. 2a, red rectangle, white arrow).
A movie was recorded by the confocal laser scanning
microscope over a 60-min period (1 frame/min) and
showed that cytokinesis failed before the formation of
a binucleated cell (Fig. 2b, white arrow, Additional file
2: Movie 1). Utilizing flow cytometry and cell count,
9.97% binucleated cells were detected by treatment
for 24 h and 0.83% in control cells (Fig. 2c, d). The
Fig. 1 Nanoparticles were internalized by cells. a: SEM photographs were 30 nm Ps particles with ultrasonic or not. Utrasonic raised the
monodispersion of 30 nm Ps particles obviously. b: Zeta potential of 30 nm Ps particels varied from -14 mV to -50 mV with ultrasonic. Potential
detector checked the Ps 30 nm particles which were negative on surface. c: 30 nm Ps particles labelled with FITC entered the cell and were
present in cytoplasm (red rectangle). d: 30 nm Ps particles gathered in large vesicle-like structures (LVLS) with time going along (white arrows)
Fig. 2 Nanoparticles induced binucleated cells formation. a: After co-culture for 12 h, the binuclear cells were present (red rectangle, white arrows)
and LVLS generated in cytoplasm. A magnified image of binucleated cell showed the 30 nm Ps particles bound on the inner surface of the LVLS.
b: Video of the process of binuclear cell formation (white arrow). c: Flow cytometer assay indicated that 30 nm Ps particles increased the percent
of binuclear cells (black line was treated cell and red line was control cell). d: The percent of binuclear cells reached 9.97 % in treated cells and
was 0.83 % in control cells. The difference of the percent of binuclear cells in treated cell and control cell was significance (p < 0.05)
rate of binucleated cells was expressed as the mean ±
standard error of the mean value determined by SEM.
The increasing rate was statistically significant
compared to the control (P < 0.05).
Influence of 30 nm Ps particles on human tumor cell lines
For detecting and confirming of the phenomenon,
A549, HePG-2 and HCT116 cell lines were selected
to repeat the test. Markedly raised percent of
binucleated cells in these treated cells(Fig. 3a,) was detected
and confirmed. Green fluorescent vesicles also
presented in the cytoplasm of these binucleated cells
(Fig. 3b, c, d). However, the rates of binucleated cells
in cancer cell lines were lower than in macrophage.
Furtherly, the rates of binucleated cells in these cell
lines treated by 30 nm Au-NPs (1.575 ng/ml) were
calculated. The rates of binucleated cells were also
higher in treated cells than control cells (Additional
file 1: Figure S4 A). Under the working dose,
statistically significant difference of viabilities of treated cells
compared to the control wasn’t detected (Additional
file 1: Figure S3 B).
Intracellular transport and distribution of the Ps
nanoparticles
For detecting the transport of the internalized
particles, we tracked existence of particle transport
vesicles in the early endosome, later endosome and
lysosome in macrophages. RAW264.7 cells were
cultured in the particle-containing medium for 10, 30
and 50 min, then rinsed by 0.01 M PBS and labelled
EEA1, Rab7 and LAMP-1(markers of early
endosome, later endosome and lysosome) with
immunofluorescence. Images showed that red fluorescence of
EEA1 and green fluorescence of particles were
colocalized and yellow spots were already present in
the cell at 10 min. After 30 min, the yellow spots
disappeared and enlarged green fluorescent flecks
present (EEA1, 30 and 50 min). In Fig. 4 (Rab7),
labels of Rab 7(red) and the particles (green) weren’t
co-localized in cells from 10 min to 50 min. The
particles weren’t transported to lysosomes either,
because the green fluorescence of particle transport
vesicles and the red fluorescence of LAMP-1 weren’t
co-localized in cells from 10 min to 50 min
(Fig. 4(LAMP-1)).
Fig. 3 Influence of 30 nm Ps particles on human tumor cells. a, binuclear A549. b, binuclear HePG-2 c: binuclear HCT116. In these binucleated
cells, vesicles with green fluorescence of Ps nanoparticles presented in cytoplasm. d: the percent of binuclear A549, HePG-2 and HCT116 cells
were 5.37 %, 7.12 % and 5.18 % in treated cells to 0.51 %, 0.63 % and 0.49 % in control respectively. The difference of the percent of binuclear
cells in treated cell and control cell was significance (p < 0.05)
Interference with membrane vesicles distribution
To visualize the recycling of membrane vesicles through
the endosomes during cell mitosis, labeled transferrin
conjugates were utilized to trace the endosomes in cells
after being treated for 12 h. Confocal fluorescent image
showed that red fluorescence flecks of transferrin
conjugates in the endosome transferred preferentially closely to
midbody region in the control cell (Fig. 5A(a)). The labels
didn’t accumulate at the midbody region in treated cell
(Fig. 5A(b)). A 3D model was constructed by acquiring a
series of Z-axis slices of the confocal fluorescent cell
image (Fig. 5B). This surface plot showed localized
fluorescence intensity of transferrin positive vesicles in the 3D
model. Compared with treated cell (Fig. 5B(b)), the
fluorescence intensity of transferrin positive vesicles at the
midbody region and the polar region of control cell was
higher(Fig. 5B(a)). 3D magnified figure shows that the
transferrin positive vesicles preferred locating close to the
LVLS (Fig. 5C, a magnified image comes from the region
of interest marked by white lines). The efflux of transferrin
conjugates from treated cells was slower compared to the
control (Fig. 5D). The time necessary for the transferrin to
return from the cytoplasm to the cell surface is delayed in
treated cells. Half-life time-points (t1/2, the time-point
when the fluorescence intensity reduced to half of the
initial value) of transferrin conjugates effluxes were
measured. The t1/2 of control cells was 2.5 min and the t1/2 of
treated cells was 4.9 min.
Expression and distribution of Rab11 and EEA1
The expressions of Rab11 and EEA1 were detected by
western blotting. Compared to the control, gray levels of
Rab11 and EEA1 bands (Fig. 6A) were increased in
treated cells with extended time of treatment (10, 30,
50 min). Labeled vesicles of Rab11 (red) and EEA1 (red)
in cell telophase were traced by confocal microscopy.
More labeled vesicles of EEA1(Fig. 6B(a)) and
Rab11(Fig. 6C(a)) with higher fluorescent intensities were
found in the midbody regions of control cell than in
treated cell (Fig. 6B(b).C(b)), which was also
confirmed by Meta Imaging Series Software (Molecular
Devices, Inc. USA) (Fig. 6D).
Fig. 4 Intracellular transport and distribution of the nanoparticles. EEA1: The co-locations (yellow) of EEA1 (red) and 30 nm Ps particles (green) were
present at 10 min, the yellow spots were magnified at right and left superior corners. The co-locations decreased at 30 min, there was hardly co-location
and the LVLS generated at 50 min. Rab7: Rab7 co-located hardly with 30 nm Ps particles at 10 min, 30 min and 50 min, the LVLS were also present in
the cell at 50 min. LAMP-1: LAMP-1 didn’t co-locate with 30 nm Ps particles from 10 min to 50 min. The co-locations of EEA1 and 30 nm Ps particles at
10 min indicated that the particles entered the cell by endocytic transport. Following that, the particles didn’t co-locate with Rab7 and LAMP-1. That
indicated that the particles were not transported through late endosome to lysosome. It meant that the 30 nm Ps particles induced the LVLS formation
in early endosome
Discussion
Viabilities of RAW264.7 cells, under varied
concentrations of Ps nanoparticles, weren’t statistically significant
compared to the control after being treated for 24 and
48 h, but the rate of binucleated cells was the highest
under the concentration of 25ug/ml for 24 h (Additional
file 1: Figure S2, A, B) (p < 0.05). A549, HePG-2 and
HCT116 cell lines treated by 30 nm Ps nanoparticles
and 30 nm Au-NPs respectively also showed statistically
significant higher rates of binucleated cells compared to
the control (Additional file 1: Figure S4(a)). Therefore,
we investigated how the nanoparticles induced
binucleated cells.
The uptake of anionic nanoparticles by cells is a
twostep process [7, 8], particles are initially bound on the
outer cell membrane, then subsequently internalized.
While binding can occur at any temperature,
internalization can’t occur at 4 °C because that the low energy level
at this temperature prevents endocytic uptake.
Confocal microscopy detected green fluorescence in the
cytoplasm of RAW264.7 cells cultured at 37 °C, but not in
those incubated at 4 °C. This suggested that most of the
particles had been internalized via endocytic pathway.
However, a previous study showed that approximately half
of the Ps particles were present in the cytoplasm even
though cells were incubated at 4 °C [9]. We have found
that the 30 nm Ps particles diluted with fresh medium
were internalized by macrophages at 4 °C if they were
treated by sonication. However, it has been observed that
the endocytic uptake of Ps particles of 50 and 100 nm
diameters was still blocked at 4 °C even after sonication.
Both dynamic light scattering (DLS) and SEM revealed
that the particles size and size distribution varied
depending on their treatment (Fig. 1a, b). Therefore, we used
ultrasonic treatment for 30 min to increase the
monodispersity of 30 nm particles, which allowed them to
directly pass through the plasma membrane [9]. The
aggregated particles were also taken into macrophages, but by
endocytosis. An unexpected result is that the particle’s
zeta potential changed as the result of sonication from
−14 mV for the aggregates to −50 mV for the well
dispersed primary particles. This implies that the aggregated
particles are unstable. It is important to note that the early
endosome lumen generally appears in the setting of
ATPdependent acidification. Under those conditions, uptake
of aggregated particles may be dispersed to the organelles.
Figure 1c shows that most particles were located in
vesicles after cells were co-cultured with particles for 1 h.
Fig. 5 Interference with membrane vesicles distribution. a: 3D-reconstruction of control cell and treated cell, red spots of transferrin were present in
control (a), red spots and green spots of Ps particles were present in treated cell (b). The red spots of transferrin accumulated at midbody in control,
but were short in treated cell. b: Surface Plot of transferrin in control (a) and treated cell (b), the fluorescence of transferrin distributed and gathered at
midbody and poles regions of control cell. But in treated cell, the transferrin dispersed in cytoplasm randomly. c. Transferrin located closely to the LVLS
contained 30 nm Ps particles (green) in 3D space. A magnified image showed the close tethers of transferrin vesicles and LVLS. d: The returning time
of transferrin from cytoplasm to cell surface in treated cell delayed obviously. The red fluorescent intensity reduction in cytoplasm of control cell was
faster than that in treated cell. The t1/2 of control cells was 2.5 min and the t1/2 of treated cells was 4.9 min. The transferrin vesicles were tethered by
LVLS contained 30 nm Ps particles
Large vesicle-like structures (LVLS, white arrow) with
green fluorescent labeled particles were also present in the
cytoplasm (Figure 1d). In the endocytic pathway, transport
vesicles pinch off pieces of the plasma membrane and fuse
with early endosomes and with incoming transport
vesicles as well as with each other. Rink et al. reported that
the formation of larger vesicles is due to homo or
heterotypic fusion of early endosomes [10]. Thus, we suggest
that LVLS are endosome components that are formed by
continuous fusion of early endosome-containing particles
with transport vesicles. Varied state of Ps particles in
endosome was a dynamic process of thermodynamic
stabilization when these particles were transported in cell.
Initial state of these particles was aggregation. Big green
fluorescent spots consisting of particles appeared in the
endosomes (Fig. 1c and d). Then these particles were
dispersed and bound on the inner membrane of endosomes
due to varied internal microenvironments (Fig. 2a). The
binding hold endosome and formed the LVLS (large
vesicle like structures).
Furtherly, binucleated cells were observed in 12 h
cultures of nanoparticle-containing medium (Fig. 2a, b).
We have recorded a video that showed how influence of
nanoparticle uptake load in macrophages and eventually
to failed cytokinesis and binucleated cell
formation(Fig. 2b, Additional file 2: Movie 1). After 24 h, flow
cytometry and cell counting revealed the presence of
9.97% and 0.83% binucleated cells in treated and in
control cells, respectively (p < 0.05) (Fig. 2c,d). Cytokinesis is
a very complex and highly orchestrated physical
separation of daughter cells. Its completion is dependent on
membrane transport and subsequent fusion in midbody
region of the cells. The localization of pivotal cytokinesis
proteins depends on the traffic of membrane vesicles
coming from endosomes. A few researchers have found
that deposition of membrane vesicles at the cleavage
Fig. 6 Expressions and distributions of Rab11 and EEA1. a: Western blotting revealed that the amount of proteins of Rab11 and EEA1 increased along
the co-culturing time (from 10 min, 30 min to 50 min). b: (a) Red fluorescent spots of Rab11 accumulated at the midbody in control cell of mitosis
telophase, (b) the red fluorescent spots accumulated to large clusters beside the midbody in treated cell of mitosis telophase. c: (a) Red fluorescent
spots of EEA1 mainly focused at the midbody in control cell of mitosis telophase, (b) the red fluorescent spots were less at the midbody
and accumulated to large clusters at the poles of treated cell of mitosis telophase. d: Fluorescence intensities of Rab11 and EEA1 at
midbody were lower obviously after co-cultured with 30 nm Ps particles. Comparted with control, the difference of Rab11 was significance (p < 0.05)
and EEA1 was without significance in treated cell. Distributions of these key regulators (EEA1 and Rab11) which regulate the organizing of contractile
ring and cytokinesis were disturbed obviously
furrow occurs during cytokinesis [11, 12]. In addition,
cytokinesis requires membrane insertion at the midbody
region to increase the cellular surface area and allow
daughter cells separation. Because plasmalemma
synthesis by the Golgi apparatus is blocked in telophase [13,
14], membrane trafficking from the endosome is the
primary plasmalemma source for cytokinesis [15, 16].
However, LVLS (marked by green fluorescence in Fig. 2a)
persisted in the cytoplasm, even when two nuclei were
visible in the cell. Although the morphology of the
binucleated cell wasn’t like apoptosis, we still assayed the
level of Annexin V conjugates in cells. Compared with
the control cells, the Annexin V conjugates in the
treated cell didn’t increase (data not shown). The result
implied that the LVLS were maintained by the particles
packaged in the structures. The LVLS may be in a
biologically inert state due to the binding of the particles,
and because there was a limited supply of membrane
components for cytokinesis, the presence of
nanoparticles prevented this process from occurring. In
induced and formed binucleated cell of A549, HePG-2
and HCT116, green fluorescent vesicles were also in
cytoplasm (Fig. 3b, c, d).
Nascent transport vesicles rapidly fuse with early
endosomes, late endosomes, and eventually with
lysosomes. We examined all three structures to delineate the
transport pathway of the internalized particles. The
tethering protein EEA1 is localized exclusively at early
endosomes and is used to target intracellular organelles [17].
EEA1 also has an important role in determining
endosome fusion efficiency [18]. We examined EEA1
redistribution by co-culturing Ps nanoparticles with RAW264.7
cells for 10 min (Fig. 4, EEA1, 10 min). In these confocal
images we found yellow spots in the cells where red
EEA1 fluorescence and green particle fluorescence
merged. After 30 min, the yellow spots disappeared, but
enlarged green fluorescent blotches were present even
though the particle containing culture medium has been
replaced with fresh medium (Fig. 4, EEA1, 50 min).
Small GTPases of the Rab family are important vesicular
transport regulators located in specific intracellular
compartments. Rab7 has been shown to localize to late
endosomes of the endocytic pathway [19]. Figure 4
shows that Rab7 (red) and the particles (green) weren’t
co-localized in cells from 10 to 50 min (Fig. 4, Rab7, 10,
30 and 50 min). LAMP-1 is a glycoprotein that is a
useful lysosomal marker [20]. The green fluorescence of
particles didn’t co-localize with the red fluorescence of
LAMP-1, indicating that the particles weren’t
transported to lysosomes (Fig. 4, LAMP1, 10 30 and 50 min).
This phenomenon was persistently in cell with time
extended to 120 min (Additional file 1: Figure S6,A,B,C).
An interesting phenomenon was that green fluorescence
of LVLS persisted for 12 h. Our research has shown that
Ps particles (d = 50 and d = 100 nm) can be transported
to lysosomes [8]. The presence of LVLS (with a clearly
outlined green fluorescence, Fig. 2a, white arrow) in
binucleated cells implied that the aggregated particles were
dispersed and bound on the inner membrane of these
endosomes. This prevented the transport of
nanoparticles from early endosomes, and the pathway from late
endosome to lysosome was also restricted. Membrane
traffic from LVLS was also blocked. Further research is
needed to clarify the detailed mechanism of this process.
There are reports that transport vesicles from the
endosome provide the source of new membrane; these
membrane vesicles are deposited in the midbody to form
a cleavage furrow and divide the cell [11, 12]. Boucrot et
al. used fluorescently labeled transferrin, a ligand that
was specifically taken up by clathrin-based endocytic
vesicles, to target membrane vesicles recycled from
endosomes during cell mitosis [11]. This method was
also utilized in our experiments to trace transferrin
vesicles in the treated cells. Confocal fluorescent microscopy
showed that vesicles with red fluorescence were
preferentially located near the midbody of control cells in
telophase (Fig. 5A(a)), but this wasn’t obvious in treated
cells (Fig. 5A(b). A reasonable hypothesis is that
internalized particles are distributed to the cell midbody
during anaphase and telophase if indeed the membrane
vesicles from LVLS participate in membrane trafficking
for cytokinesis. Figure 5A(b) shows that treated cells
lacked green fluorescence in the midbody region in
telophase, whereas red labels of transferrin conjugates were
also randomly distributed in the cytoplasm of treated
cells. Schweitzer et al. reported that transferrin vesicles
were transported from the distal endosomal cluster to
the intercellular midbody in late mitosis [21]. In our
experiment, the phenomenon was also present in control
cell (Fig. 5A(b)). Under 3D high magnification, these
transferrin-positive vesicles in treated cells weren’t
proximal to the midbody, rather, they were near the LVLS
(Fig. 5C, A magnified image comes from the region of
interest marked by white line). The efflux of transferrin
conjugates from the treated cell was slower compared to
the control (Fig. 5D). The time for transferrin returning
back to the surface membrane of the cell was delayed in
treated cells (the t1/2 of control cells was 2.5 min and
the t1/2 of treated cells was 4.9 min). This result implies
that the LVLS tether these transferrin-positive vesicles
and disturbs the membrane vesicle transport of
transferrin during telophase (Additional file 1: Figure S5).
Endosomes transport of Rab11 to the midbody of
mitotic cells is necessary for establishing the abscission site.
During this process, Rab11 GTPase binds and activates
various effector proteins involved in targeting vesicles
during mitosis. EEA1 is localized exclusively to early
endosomes [18], where it acts as a tethering molecule that
couples vesicle docking with SNARE proteins to bring the
endosomes physically closer and facilitate their fusion. We
assessed Rab11 and EEA1 expressions by western blotting
and found that both Rab11 and EEA1 (Fig. 6A) were
increased in treated macrophages. Confocal fluorescent
microscopy revealed the distributions of these cells with
Rab11 and EEA1 label (red) in telophase, there were fewer
labeled vesicles were found in the midbody region of
treated cells (Fig. 6B(b),6C(b)) compared with control
cells. Thus, internalized particles induced high Rab11 and
EEA1 expressions, but LVLS with particles disturbed their
distributions. Accurate Rab11 localization is critical for
the execution of cytokinesis. A series of studies revealed
that Rab11-binding proteins FIP3 and FIP4 located to the
midbody and simultaneously interacted with Rab11 to
promote cytokinesis [22–24]. A reasonable suggestion is
that LVLS in nanoparticle-treated cells altered Rab 11 and
EEA1 localizations.
The research article assessed the influences of 30 nm
Ps particles and 30 nm Au-NPs on the endocytic
pathway in RAW264.7, A549, HePG-2, and HCT116 cell
lines, through the formation of LVLS, both
macrophage(RAW264.7) and human cancer cell lines(A549,
HePG-2, HCT116) resulted in increasing percent of
binucleated cells. Many papers have reported the
formation of binucleated cells by interfering in the endocytic
pathway, but the process of internalization of
nanoparticles (30 nm Ps particle and 30 nm Au-NPs) through
endocytic pathway could lead to the same effect has not
been reported intensively. So our work described a new
phenomenon of nanoparticles effect on endocytic
pathway in different cell lines. On the other side, all of the
data supporting our opinion was collected on cell level
only and under our laboratory conditions.
aggregated particles disperse and bind on the inner
endosome membrane depending on the
microenvironment. The LVLS block membrane trafficking from the
LVLS and the endosome, limit the amount of membrane
components available, and alter the localization of
pivotal proteins required for cytokinesis. Importantly, we
have found that nanoparticle-treated cells have difficulty
completing this important process because of limited
membrane trafficking.
Additional file 1: Supplemental data. (DOCX 22634 kb)
Additional file 2: Movie of failing cytokinesis. (MP4 5 kb)
Abbreviations
BCA: Bicinchoninic acid; BSA: Bovine serum albumin; CCK-8: Cell counting kit-8;
DMEM: Dulbecco’s minimal essential media; EEA1: Early endosomal antigen-1;
FBS: Fetal bovine serum; LVLS: Large vesicle-like structures; Rab: Ras-related
protein; TBST: Tris-buffered saline and tween 20
Acknowledgements
This work was supported by Key laboratory of Biological effects of
Nanomaterials and Nanosafety, Chinese Academy of Sciences.
Authors’ contributions
XL CYN and ZMY performed experiments, XL collected data and wrote the
manuscript; GWH CK YL and BX assessed the Ps and Au NPs and performed
experiments. LJ analyzed the data; LS and XGM contributed to the
experiment design. All authors read and approved the final manuscript.
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