The effects of long term exposure of silica nanoparticles
to RAT-1 fibroblast cells.
Abstract:
Nowadays nanoparticles are already used in a lot of products. In some situations humans and animals are
exposed to nanoparticles daily. Despite the widespread use, there is an important question to be answered:
What are exactly the risks of nanoparticles to human health? A lot of research is already done, but still some
answers are missing. Studies on cultured cells might help answering this question. In this article the effects of
two concentrations (0.01 mg/ml and 0.1 mg/ml) Silica nanoparticles to RAT-1 fibroblast cells is studied. The
main goals are to study the effects of the nanoparticles directly after exposure and after longer periods.
Therefore RAT-1 fibroblast cells were exposed to the silica nanoparticles and followed during three weeks with
light and electron microscopy. A second very interesting point are the effects of a much smaller dose of nanoparticles exposure of the cells for longer periods. In some daily used products nanoparticles are already added.
What are the effects of those low daily concentrations to the cell? Again the effects of those low doses are
studied with light and electron microscopy.
This article demonstrates that as soon the nanoparticles are added to the cells the viability of the cells drops
down. This effect is dose depended. During the three weeks the viability increases slowly. The cells are recovering and at the end of the three weeks they appeared almost normal, except the nanoparticles still inside the
cells. They accumulate inside the vesicles. Even a regular low dose will accumulate and will disturb the cell the
same as a high dose of nanoparticles. This indicates that even a very low concentration of nanoparticles could
be a problem: it accumulates.
Introduction
Everywhere on earth, nanoparticles are created by natural processes or by humans processes, for example
industrial manufacturing and nanotechnology1,2. Humans and other organisms can be exposed to these nanoparticles, for example by inhalation. The way of internalization and the toxicological effects in the cells depends on the unique properties of the nanoparticles2. One of those properties is the size. Because of the size
nanoparticles will react different with cells than bulk material. Other unique properties are the chemical composition, surface structure, shape, aggregation and concentration 2-4.
Effects in the body
As soon the nanoparticles enter into the body different processes will be activated or disturbed. If nanoparticles are inhaled by the lungs, lung fibrosis could arise2. In the lung the nanoparticles will also activate the
body’s defence mechanisms 5. This defence mechanism tries to clear the nanoparticles from the body, especially when the body is exposed to a high concentration of nanoparticles5. A part of this defence mechanism
are the immune cells2. The activation of immune cells creates products, for example cytokines, which will have
a negative influence on other surrounding cells6. Not all the nanoparticles will be cleared by these mechanisms. The rest will be transported via the bloodstream through the whole body, whereby the nanoparticles
can reach every cell.
In the blood the nanoparticles can affect the coagulation, which causes platelet aggregation and the formation
of microtrombi, but it can also activate haemolysis, thrombosis and systemic inflammation2, 7, 8. The particles
are transported to the liver, which can causes hepatotoxicity through activation of a large number of phagocytic cell2, or to the gastrointestinal system, where the epithelial cells of the intestines will be affected 9. The particles can also be internalized by nerve cells, which causes neurodegenerative diseases2. When the nanoparticles are internalized by the trigeminus nerve and the tracheobronchial sensory nerves, the nanoparticles will
have an indirect effect on the cardiovascular system8. This are only a few examples of organ systems affected
by the nanoparticles. Because of the size of the nanoparticles they can reach almost every organ system, so all
the systems could in principle be affected by exposure to nanoparticles.
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Effects on cell level
On cell level the nanoparticles can have different effects. It depends on the type of cell which effects the exposure to nanoparticle will have10. Despite, a distinction can be made between two categories of cells, namely
phagocytic cells and non-phagocytic cells.
Phagocytic cells
As already mentioned the nanoparticles will activated the defence mechanism of the body. Kulikova et al discovered that only the macrophages will be activated and not the lymphocytes11. This is because specialized
receptors are needed6. The macrophages have those receptors because the innate cells will contact the nanoparticles, whereby these cells can react with the antigens on the nanoparticles. After that the macrophage will
have the right receptor to internalize the nanoparticles 11. The cells can internalize the nanoparticles by different processes. For example, the macrophages and other phagocytic cells will internalize the nanoparticles by
phagocytosis. Phagocytosis is a clathrin dependent endocytic pathway 6, where specific cell-surface receptors,
such as the scavenger-A receptors, are needed1,12. Orr et al suggested in their article that this receptor can also
bind nanoparticles6. Macrophages can also internalize the smaller nanoparticles by patocytosis. This is an additional endocytic pathway, whereby the nanoparticles are transported through induced plasma membrane
channels into an extensive labyrinth of interconnected membrane-bound compartments13.
In the macrophages the nanoparticles are accumulated inside the endosomes and lysosomes. Lysosomes are
trying to degrade the nanoparticles, however due to the nature of the material the lysosomal enzymes are in
general not capable of degrading the nanoparticles, which might result in intracellular accumulation. Lucaini et
al found that the accumulation of nanoparticles causes activation of a mechanism called microvasiculation.
Microvasiculation is the process whereby small vesicles (100-1000nm) are shed from the plasma membrane to
the external environment, with inside the nanoparticles. This might be a mechanism by which nanoparticles
are removed from cells. These vesicles can be internalized by other macrophages14.
Non-phagocytic cells
Non-phagocytic cells can also internalize the nanoparticles. The precise mechanisms by which the nanoparticles are internalized are unknown. Several pathways have been reported as possible internalization mechanisms. Most researchers agreed that endocytosis is the mechanism of internalization 2, 6, 12. But endocytosis
can be separated into 4 basic mechanisms: macropinocytosis, clathrin-mediated endocytosis, caveolaemediated endocytosis and the clathrin- and caveolae-independent endocytosis 12. By which mechanism of
endocytosis the nanoparticles are internalized is not clear. In the literature two types of mechanisms, the
clatherin mediated and the caveolae mediated pathways, are proposed to be the most likely pathways of internalizing the nanoparticles 14.
Some researches do not agree with endocytosis as mechanism. For example, Geiser et al found that polystyrene microspheres (0.2 and 0.078 µm) were internalized in a “non-membrane surrounded” way4. This indicates
a non-endocytic uptake mechanism, for example diffusion. The nanoparticles were present in the cytosol
without a surrounding membrane. It is possible that nanoparticles smaller than 50 nm are going into the cell
by diffusion through the integral membrane protein pumps or channels4. Another mechanism was discovered
by Le Bihan et al. They found nanoparticles inside vesicles, surrounded by a lipid bilayer. They concluded that
this lipid bilayer belongs to liposomes. With the help of these liposomes nanoparticles can be transported into
the cells via an invagination process without the need of supplementary energy 15. Not all the nanoparticles can
be internalized by liposomes. Only nanoparticles larger than 30 nm are able to form a total adhesion energy
larger than the energy costs associated with the bending of the bilayer and with that they can go through the
bilayer. Particles smaller than 30nm were found outside those vesicles, bound to the outer surface of the liposome15. After internalization the nanoparticles will accumulate inside the cell. It is suggested that nonphagocytic cells used a comparable mechanism as microvasiculation in the phagocytic cells, to remove nanoparticles from the cytosol, but this is still unknown14.
The nanoparticles will influence the cell processes. The effects will vary from cell type to cell type, because of
cell type specific factors as metabolism10. But all cell types will show signs of cellular stress16 . In figure 1 the
effects of nanoparticles in cells are summarized25. The main processes which are affected by the nanoparticles
are the protein synthesis, the metabolism of the cell and the nucleus with DNA. Of course the cell will also
react with an inflammatory response, by release of cytokines and other mediators. These processes together
can activate cellular stress, apoptotic processes and finally will cause cell death. All the different types of cells
will react on the nanoparticles by one or several of these processes. Which processes depends on the type of
cell.
2
Oxidative stressassociated proteins
Cytoskeletonassociated proteins
molecular
chaperones
(Regulation of
transcription)
Protein synthesis
Energy metabolism
associated proteins
Oxidative stress
Apoptosis
energy problems in
mitochondria
(Metabolism)
Redox active
intermediates
Tumor associated
proteins
Photo exication
Cell
DNA damage
Cell cycle arrest
Nucleus
Mutagenesis
Inflammatory
response
Apoptosis
Figure 1:The possible effects of nanoparticles in cells. Not all the reactions will occur as a cell is exposed to nanoparticles. Depending on the type of nanoparticle and cell, different reactions can be activated 25
Some questions still unanswered
Until now nanoparticles are studied frequently. A lot of information is already known about the properties and
effects of nanoparticles on cells, but still there is a lot of missing information. Especially for non-phagocytic
cells the process of internalization is unclear, even as the long term effects of a single exposure to the nanoparticles and also the long term exposure of nanoparticles to cells. In this paper these questions will addressed.
Because every type of nanomaterial, every size and every cell type will react different to each other, first the
ideal dose must be determined for silica nanoparticles in RAT-1 fibroblast cells. It is important to know which
nanoparticles can be used and what concentration for the best results. Furthermore it is important to know
what the effects of these nanoparticles are at short exposure of nanoparticles to this type of cell. For this reason a dose response experiment was done with two types of silica nanoparticles (F200 or F380). Using light
and electron microscopy the effects of these nanoparticles in RAT-1 fibroblast cells was determined.
As already mentioned a lot of research was done to discover the internalization processes. Still there are some
questions unanswered. One of those questions is the mechanism of internalization of the nanoparticles in nonphagocytic cells. Several mechanisms are possible but which one is used is unknown. The use of immunofluorescence might allow us to distinguish between different mechanisms and discover by which mechanism the
3
nanoparticles are internalized in RAT-1 fibroblast cells.
Another important question is the long term effects of the nanoparticles. Until now only research was done to
discover the effect of the nanoparticles on the moment the nanoparticles were introduced to the cells or tissue. The effects of the nanoparticles when they are inside the cells or tissue after a month is not yet examined,
neither are the effects of long term exposure to the nanoparticles. Hildebrand et al found in their experiment
that at certain concentrations of nanoparticles cell viability was almost recovered after 3 days 16. They also
concluded that it is not easy to predict long-term consequences of nanoparticles exposure or accumulation. In
this research these questions will be answered using light and electron microscopy.
Material and Methods
Nanoparticles and characterization: Two types of nanoparticles are used, the 200F and the 380F. Both the
particles are hydrophilic fumed silica. The difference in properties between both kinds of nanoparticles are
given in table 1 (http://www.aerosil.com). The main difference is the surface area. Following table 1 the surface area of F380 nanoparticles is larger than the surface area of F200 nanoparticles.
Properties
Specific surface area (BET) = m2/g
Tamped density (acc. To DIN EN ISO 787/11, Aug. 1983= g/l
Moisture (2 hours at 105⁰C)= wt.%
Ignition loss (2 hours at 1000⁰C based on material dried for 2 hours at
105⁰C)= wt.%
pH (in 4% dispersion)
SiO2 – content (based on ignited material) = wt.%
F200
200 ± 25
Approx.
50
<1.5
<1.0
F380
380 ±30
Approx.
50
<2.0
<2.5
3.7-4.7
>99.8
3.7-4.7
>99.8
Table 1: The main properties of both nanoparticle types (www. aerosil.com)
Dose response relation:
Cell culture and exposure of cells to nanoparticles: The RAT-1 fibroblast cells were kept in cell culture medium
at 37⁰C. Culture medium consisted of: DMEM 1* (Dulbecco's modified eagle medium) including 4.5g/l glucose
and NEAA, but without L-glutamine and Pyruvate (firma Invitrogen). To 500ml medium are added 45ml FCS
(foetaal calf serum; firma PAA) and 5 ml pen/strep/glut (+10000Units/ml pen + 10000ug/ml strep +29.2 mg/ml
L-glut; firma Invitrogen). For the experiment they were cultured in the wells of a 6 wells plate. To reduce variability in the results cells were always used at the same passage number. They were plated at a density of 4 x104
cells/cm2 and allowed to attach and grow for 3 days.
The nanoparticles were diluted in MQ by sonification for ¾ hour (1 mg/ml). Every well with fibroblast cells was
filled with medium and the diluted nanoparticles in the concentrations: 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml,
0.0625 mg/ml & 0.03125 mg/ml. These concentrations were made by adding culture medium to the diluted
nanoparticles. For both of the nanoparticles a control (0 mg/ml particles) was made with only the maximum
amount MQ. The cells were incubated for 24 hours at 37⁰C. After the incubation the cells were fixated with a
Karnovsky fixative. For 1 hour at RT the fibroblasts cells were immersed in the aldehyde-based fixative existing
of 2% formaldehyde, 2.5% glutaraldehyde in 0.08M sodium-cacodylate buffer (pH 7.4) containing 0,25 mM
CaCl2 and 0,5 mM Mg Cl2.
Microscopy: First the condition of the cells under different conditions was checked with the light microscope.
The wells in which part of the cells were still alive and attached to the substrate could be used for further research and were prepared for the electron microscope.
The wells (with Karnovsky) were washed thoroughly with CaCo (0,1 M sodium-cacodylate buffer) two times
and then incubated with a post-fixative solution of 1% OsO4 (EMS) + 1.5% Potassium Ferro cyanide
(K4[Fe(CN)6]) in 0.1M cacodylate buffer (CaCo) for 2 hours, whereby the cells were kept on ice in the dark. After
washing the cells thoroughly with distilled water the dehydration was started with ethanol percentages of
70,80,90, 96 and 100%. Then an epon mix (half epon (Fluka, Steinheim, Germany); half 100% ethanol) was
added to the wells. The well-plates were left overnight on a moving platform and the next day the epon mix
was changed for a new epon mix (1 part 100% ethanol and 2 parts epon). And at the end of the day again the
epon mix was changed for another one (1 part 100% ethanol and 3 parts epon). Again the well-plates were left
overnight on a moving platform and next day the epon mix was changed for pure epon. After a couple of
4
hours another time new fresh pure epon was added and in every well capsules were put on top of the cells in
the small layer of epon thus making the samples. After one night the small layer of epon in every capsule was
polymerized, all the malls were filled completely with epon and a paper identification label was added. After
another 48 hours of polymerization the samples were broken from the bottom of the well and oblique sections
(50 nm for TEM) were cut with a microtome with an angle of 10 degrees to the cells monolayer. The sections
were collected on Cupper EM Grids coated with formvar films and carbon. The grids were contrasted with 7%
uranylacetate (in70% methanol) and leadcitrate (according to Reynolds in distilled water). The samples were
imaged by electron microscopy using a FEI Tecnai 10 and 12.
Uptake mechanism:
Cell culture and exposure of cells to nanoparticles: The study of the uptake mechanism was done in two different parts. First, using EM the uptake of nanoparticles was studied the first 4 hours after nanoparticles were
added to the cells. For this experiment the same protocol was used as for the dose response relationship.
There are only a few differences. The cells were not cultured in a 6 wells plate, but in small petridishes (3 cm2).
This was done because every petridish was incubated for a different time with nanoparticles: 0 hours, 4 hours,
1 day and 2 days. Another difference is that only 3 concentrations were used: 0.1 mg/ml, 0.01 mg/ml and 0
mg/ml. These cells were also fixated with Karnovsky (see dose response), but the moment of fixation differs
for each sample. The samples were kept in Karnovsky fixative until all samples were collected. Only the last
sample was fixated for 1 hour.
Microscopy: The preparation of the samples for electron microscopy was done exactly the same way as the
preparation for the dose response experiment.
For studying the uptake mechanism also fluorescence microscopy was used. Based on literature nanoparticles
of 7 nm can be internalized by the clathrin-depended pathway or by the caveolin-depend pathway (see introduction). For this reason the following antibodies were chosen: LAMP1 (lysosome; BD Biosciences, 555798),
LAMP2 (lysosome; BD Biosciences, 555803), Caveolin (caveola;BD biosciences, 610059 ), EEA-1 ( endosome; BD
Biosciences, 610457), Clathrin (clatherin coated pit; BD Biosciences, 610499), CD63 (lysosome; BD Biosciences,
551458). Also lysotracker was used. For all the different antibodies in combination with the RAT-1 fibroblast
cells the ideal concentration is unknown, so before starting the real experiment different concentrations had
to be tested.
The cells were grown in petridishes which contain aclar pieces with a matrigel coating. In de beginning of the
immunofluorescence protocol, first the aclar pieces were cut with a blade in smaller pieces. This was done
after the cells where fixated for 15 minutes with 1% PFA in 0,1 M PHEM-buffer (pH=6.9) and after the first two
washing steps with PBS. Then the cells were washed again with PBS. Permeabillisation of the cells was done
for 10 minutes in PBS/0.1% saponin and after that the aldehyde groups were quenched with 100mM NH4CL in
PBS/0.1% saponin for 10 minutes. Before the primary antibody was added the cells were washed again 3 times
2 minutes with PBS/0.1% saponin. Cells were incubated for 1 hour in a moisture chamber with primary antibodies, diluted in PBS/0.1% saponin/1% BSA. After 1 hour the cells were washed again 3 times 10 minutes with
PBS/0.1% saponin and the secondary antibody, diluted in PBS/0.1% saponin/1% BSA, was added for 1 hour in a
dark moisture chamber. After this step the samples were kept in a dark environment. First the samples were
washed 3 times 10 minutes with PBS/0.1% saponin and then DAPI staining was started: incubation of DAPI
1:500 in PBS/0.1% saponin for 5 minutes in a dark moisture chamber. After washing 2 times 5 minutes with
PBS and 2 times briefly with aquadest the excess of water was absorbed with care onto filterpaper. Now the
aclar pieces were ready to mount with 7 µl prolong gold on a glass slide. After 24 hours the result can be seen
inside the immunofluorescence microscope.
For some samples the lysotracker was used. The lysotracker was added for 1 hour at 37®C before the fixation
of the cells started. Then the protocol written above was done exactly the same way, except all steps were in
the dark now.
Long term effects:
Cell culture and exposure of cells to nanoparticles: Before the cells were cultured in 3 cm2 petridishes, two
aclar pieces were pasted with small droplets of matrigel inside the dishes. These dishes with aclar pieces were
coated with matrigel. The aclar pieces were used to make it possible to collect samples on every moment
without losing a complete dish. The cells growing on the bottom of the dishes are used for the SRB-assay.
Matrigel was needed to be sure the cells were still attached to the bottom of the dish and the aclar. For every
interesting time moment one petridish was used. To reduce variability in the results cells were always used at
the same passage number, in this case P2. Again the cells were plated at a density of 4 x104 cells/cm2 and al-
5
lowed to attach and grow for 3 days before the nanoparticles are added. Every dish with fibroblast cells was
filled with medium and the diluted nanoparticles in the concentrations: 0.1; 0.01 mg/ml or the control (0
mg/ml) particles.
After the incubation of the nanoparticles to the cells for an certain time moment, the aclar pieces were removed from the petridishes and were collected in a 12 well plate with in the well 1 ml of the Karnovsky fixative
(see dose response experiment). The pieces stayed in the fixative until all the samples of that experiment were
collected.
Microscopy: The protocol is almost the same as the procotol described by the dose response experiment until
the cells were kept inside pure epon. After this step the aclar pieces were cut with a blade in small pieces.
These pieces were embedded in two different ways. A part of the pieces was embedded in special malls,
whereby the pieces were embedded in the length of the epon (laying on the bottom of the malls) and also a
paper identification label was added. The other part of the pieces were imbedded laid on the bottom of a
petridish in a small layer of epon, whereby little capsules were put on the top of the aclar pieces. After one
night the samples were polymerized and for the pieces in the petridish the little capsules were filled with a
paper identification label and epon.
After complete polymerization (48 hrs) the samples were taken from the malls and sections (50 nm for TEM)
were cut. For the aclar pieces (laying on the bottom of the malls) the cells were cut with the microtome normally perpendicular (90 degrees to the cell layers). For the aclar pieces in a capsule the capsule was removed
and the cells were cut with the microtome the same as the uptake mechanism experiment: 10 degrees to the
cells layer. The sections were collected and handled the same as above.
SRB-assay (sulforhodamine B):
To determine the number of cell still available, the SRB-assay was done. With this assay the cell density was
determined by measurement of the cellular protein content 17. After the aclar pieces were removed from the
dishes, the medium was removed and 1 ml cold 10% trichloroacetic acid was added to the dishes for 1 hour at
7 degrees, to fixate the cells. Then the dishes were washed repeatedly with aquadest and SRB (0.4 mg sulforhodamine/ ml 1% acetic acid) was added for 30 minutes. By washing the plates with 1% acetic acid the
excess dye was removed and 10mM Tris base solution(pH=7.5) was added whereby the protein bound dye was
dissolved. From every dish 4x 100 µl was added to a 96-wells plate, which was measured by a microplate
reader at OD 570 nm.
Results
Dose response relation
Before the effects of nanoparticles on RAT-1 fibroblast cells can be tested, the optimal concentration of nanoparticles had to be determined. This was done for 2 different silica nanoparticles: F200 and F380 nanoparticles.
The nanoparticles were added in different concentrations to RAT-1 fibroblast cells for 24 hours. In figure 2 the
LM pictures are shown: This figure shows that both type of nanoparticles affect the cells. Cells exposed to
nanoparticles looks different compared to the control cells, for example picture yellow C and D compared to
yellow A (cells exposed to F200 nanoparticles). Especially at higher concentrations cells are more and more
affected and death cells are visible. This can be seen in, for example, figure 2, picture red E and F where the
structure of the cells is irregular instead of the normal cell structures in the control. The exposure to the different types of nanoparticles shows a different reaction. As seen in the figure, cells exposed to F380 particles
will be affected only at higher concentrations while the cells exposed to F200 nanoparticles are affected earlier
and more heavy. The pictures of the highest concentrations of F200 (Picture yellow E and F) shows almost no
cells. Probably the cells which are visible are also dead or almost dead. Based on these results, the F200 nanoparticles are more toxic to the cells than the F380 nanoparticles.
6
Figure 2: LM pictures of the dose-response-experiment with F380 and F200 nanoparticles in RAT-1 fibroblast cells.
The different types of nanoparticles causes different effects in the cell. F200 (yellow letters) causes more damage
and cell death than the F380 (red letters). Every letter indicates a different concentration, as noted inside the middle of the figure. Especially at high concentration a lot of cell loss is visible (pictures F).
After light microscopy imaging the same cells were prepared for the electron microscope. In figure 3 the different effects of both types of nanoparticles at different concentrations after exposure of 24 hours are shown.
Again a lot of affected cells were visible after the nanoparticles were added. There is also a difference between the two types of nanoparticles visible. The F200 nanoparticles affect the cells more than the F380 nanoparticles. Cells exposed to F200 are more damaged and are affected at lower concentrations than the F380
nanoparticles This can be seen as large vesicles in the cell, as shown in picture A and B of figure 3.
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Figure 3: EM pictures of dose response experiment with F200 and F380 nanoparticles in RAT-1 fibroblast cells. The pictures are clustered together: the second picture is a close up of the first one. For example picture B is a part of picture A
(inside the square) but at higher magnification. Both types of nanoparticles causes cell damage: Cells exposed to F200
nanoparticles (A-F) have a lot of vesicles, while the cells exposed to the F380 nanoparticles have less vesicles and damage, except at the highest concentrations. The highest concentrations of F200 didn’t have any cells or cell structures left,
so they weren’t studied with EM.
8
Also a lot of nanoparticles can be found inside the cells, see picture C and D figure 3. For cells exposed to F200
nanoparticles, affected or death cells can be found in every concentration, even the lowest. For the F380 nanoparticles the cells exposed to the lowest concentrations only show cells which are a little affected. There are
nanoparticles inside those cells, but the cell structure looks the same as in normal, healthy cells. Only the highest concentration of F380 shows severely affected cells, while the highest concentration of F200 was not used
for EM since no cells were detectable under the light microscope. The results of the dose response experiment
point out that it is best to use F380 nanoparticles for the next experiments. This is because the cells exposed to
F380 still have recognizable cell structures and cells probably still function normally or almost normally. For
the next experiments only two concentrations of nanoparticles were used. The highest concentration was 0.1
mg/ml because in the pre-experiment the concentration 0.125mg/ml showed only little affected cells. Many
nanoparticles can be found in those cells, but the structures are still recognizable. The lowest concentration in
further experiments was 0.01 mg/ml, because in the pre-experiment the lowest concentration 0.03125 mg/ml
didn’t show affected cells, but did show uptake. So a lower concentration will not affect the cells.
Before the other experiments started one thing had to be checked. Are the particles visible in EM in the cells
really the F380 nanoparticles? Based on the results found in the dose response experiment it is difficult identify the nanoparticles in the cells with 100% certainty because there are more structures which looks like the
particles. To distinguish between the different particles an element analysis was done (figure 4). In this figure
the elements inside the white dot (a vesicle suspected to be filled with nanoparticles; right in the photo (arrow)) are measured. In the graph can be seen that the white dot exists of Oxygen (OK light blue) and Silica
(SiK  pink & SiM  green). These elements are available the most. So the white dot in this picture is a cluster
of F380 silica nanoparticles, the intensity of this element is the highest.
intensity (arbitary units)
Element analysis F380
Nanoparticle
18.00
16.00
14.00
12.00
10.00
8.00
6.00
4.00
2.00
0.00
PbM
PbL
SiM
OsL
OK
UM
UL
location (10nm)
SiK
Figure 4: EDX analysis of F380 nanoparticles to be sure silica nanoparticles are visible inside those vesicles . The light
blue line indicates silica nanoparticles. Around a location of 140 nm silica is the most available of all the elements.
This is on the white dot in the vesicle (see arrow). This proves that the white dot is a cluster of silica nanoparticles.
Uptake mechanism
The uptake mechanism was studied first by immunofluorescence. The cells used in the experiment are RAT-1
fibroblast cells. It was difficult to find literature about the best concentration of the antibodies for this type of
cell. Therefore a lot of pre-experiments were done to determine the best antibodies and concentrations for
the different structures of interest. In these pre-experiments suitable antibodies to lysosomes, endosomes,
clathrin and caveolin were found. The best results were found with the following concentrations:
Lysosomes: CD63 in a higher concentration than 1:150
Clathrin coated pits: Clathrin in a concentration of 1:50
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Caveaolae: Caveolin in a concentration of 1:300
EEA-1 for endosomes, in a concentration of 1:500
Also lysotracker can be used to find the lysosomes inside the fibroblast cells.
Despite of these results the study of the uptake mechanism by immunofluorescence was not done. This because even the best antibodies and concentration caused a lot of background signals. It was difficult to distinguish between structure of interest and a cluster of cell membrane proteins. For this reason this experiment
was finally cancelled.
Another possible way to study by which uptake mechanism the cells internalizes the nanoparticles is by determining the
moment the first nanoparticles were internalized inside the cell. For that reason cells were exposed to F380
nanoparticles for 10 minutes, 30 minutes, 1 hour and 2 hours and studied by EM. The results are shown in
figure 5. Especially the first 30 minutes of the highest concentration are important, because cells exposed to
the highest concentration are already affected after 30 minutes. On that moment nanoparticles must have
entered the cells, otherwise cells will not be affected this way. In the samples of 10 minutes (highest concentration) only in a single cell the internalization process has already started. In most of the cells the nanoparticles are internalized between 10 and 30 minutes. For the lowest concentration (0.01 mg/ml) it was difficult to
see the nanoparticles in the cells. Only in the cells exposed for 2 hours to the nanoparticles at 0.01 mg/ml, the
nanoparticles were found inside the cell. In the picture of 1 hour some nanoparticles are being internalized
inside the cell (left, above in the picture). Except nanoparticles enter the cells, the cells must have a way to
excrete the nanoparticles. In figure 5 can be seen that after two hours (0.1 mg/ml) the cell probably excretes
the internalized nanoparticles by the process of exocytosis. The nanoparticles are found outside the cell between some cell rests. Those cell rests can also indicate that the cell is dying and in the process of dying the
cell lyses and nanoparticles are released into the extracellular environment.
Long term effects
The effects of the nanoparticles were measured after varying periods during the first 3 weeks after exposure.
Two different groups were studied: The effects on the cell after a short exposure: only once nanoparticles are
added (A in figure 6). This was done for the concentrations 0.01 and 0.1 mg/ml. In the other group (B in figure
6) cells were exposed twice a week to new fresh nanoparticles. This was only done for the concentration 0.01
mg/ml. Both groups are tested in the same experiment, where the other circumstances are exactly the same,
so the control (0 mg/ml) can be used for both groups. In figure 6 a systematic overview of the three different
approaches are shown. Comparable to the dose response experiment the cells were first studied with light
microscopy to be sure of the presence of the cells inside the petridish and to monitor if the effects of the nanoparticles on the cells are already visible. After that some samples were used for electron microscopy. Only a
few samples were prepared for EM. For choosing the right samples, first a viability test is done.
A
3 weeks
Figure 6: An overview of the different ways the effect of the nanoparticles are tested. The red lines indicate the moments the nanoparticles are added, while the green lines indicate the moments the effects are measured by LM and EM.
This figure is only an indication of the three different ways, not the real moments of adding and measuring are shown.
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Figure 5: EM pictures of the first 2 hours of RAT-1 fibroblast cells exposed to different concentrations of F380 nanoparticles. The
left column are cells not exposed to the nanoparticles. These already have a lot of vesicles inside the cell. The middle column are
the cells exposed to the lowest concentration (0.01 mg/ml) of nanoparticles. Especially after 2 hours nanoparticles are visible
inside the cell. The right column are pictures of cells exposed to the highest concentration (0.1 mg/ml). After 30 minutes the first
nanoparticles are internalized. This concentration causes a lot of damage, see for example 2 hours.
11
Short exposure
In figure 7 the light microscopic results of cells exposed to the concentration of 0.1 mg/ml nanoparticles for
different durations are shown. In picture A of this figure the cells are shown before nanoparticles were added.
This is the control. These cells look normal. As soon nanoparticles were added to the cells, more and more
affected and dead cells can be found. In picture B a lot of vesicles are visible inside the cells. This is an
indication of apoptotic cells. The completely dead cells are visible as white dots, in picture C-G. These
dettached from the bottom and are floating in the medium. After 3 days some recovery can be seen, because
there are a lot of dead and affected cells, but also also some normal, healthy cells visible. In picture E and F
recovery can be observed by the presence of normal cells between the affected cells. The recovery continued
for 2 weeks (E-I). After 2.5 weeks the cells look as normal, compared with the control (J).
Also the concentration 0.01 mg/ml was photographed with light microscopy (results not given). The cells did
react at the moment the nanoparticles were added, but not as extreme as the cells did which are exposed to
0.1 mg/ml nanoparticles. There was only a small effect visible because some cells are affected and dying
because of the nanoparticles.
From the light microscopic pictures shown in figure 7 can be concluded that the cell layer will recover, because
at the end, after 2.5 weeks, the cell layer look almost the same as the control picture. So some cells are able to
survive the nanoparticles, even at high concentrations. Only these cells will proliferate again and recover the
cell layer. If the cells themselves will completely recover is difficult to determine based on light microscopic
samples.Therefore some samples are prepared for electron microscopy, based on the viability of the cells after
exposure. Therefore first a viability test was done.
To determine the viability (number) of the cells the SRB-assay was used. In figure 8 the results of this assay are
shown. As soon the nanoparticles were added to the cells and they were internalized into the cell, the cell
number drops. Especially when the high concentration (0.1 mg/ml) of nanoparticles was used. This is given by
the blue line. The lower concentrations of nanoparticles cause a small decrease in the viability, but this is recovered very soon (day 2, redline). Also the decrease of viability of the high concentration will recover, but as
shown in the figure it takes a week to recover.
Viability of fibroblast cells exposed to F380 nanoparticles
3
2.5
OD 570
2
0,1 mg/ml short
1.5
0,01 mg/ml short
1
0,01 mg/ml long
control
0.5
0
3 weeks
2 weeks
1 week
4 day
3 day
2 day
1 day
4h
0h
Figure 8: Viability of Rat-1 fibroblast cells exposed to F380 nanoparticles the first 3 weeks after exposure. The purple line is the
control line. During the 3 weeks the viability of the control cells increased, especially at the end. The green and red line are
both the concentration 0.01 mg/ml. The red (short) line is only exposed for 4 hours to the nanoparticles, while the green is
exposed 2 times a week to the nanoparticles. Both the circumstances have almost the same viability. The highest concentration (blue) decreased the viability of the cells, but recovers after a week. N=1.
Following these results four moments for EM sample preparation were chosen, namely 4 hours, 2 days, 4 days
and 3 weeks. After four hours the lines show a drop in viability, so probably a lot of cells are affected on that
moment. After 2 days the cells exposed to the lowest concentration are recovered, while the cells exposed to
the highest concentration are on their lowest viability. The moment of 4 days is chosen because on this moment even the cells with the highest concentration are almost recovered. Of course the last moment could
also be interesting to study the effect of recovering.
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Figure 7: LM pictures of Rat-1 fibroblast cells exposed to 0.1 mg/ml F380 nanoparticles during the first 3 weeks. A couple hours
after adding nanoparticles to the cells, the cells are affected and most of them die in the first days. Around a week after exposure
to nanoparticles the cell population started to recover and after 2 weeks of exposure the cell population has recovered.
13
In the already partly described figure 5 and in figure 9 the results of the electron microscopy are shown. In
figure 5 the effects of the nanoparticles can be determined just after exposure to the particles. In this figure
only three different concentrations are shown. The left column shows the cells not exposed to the nanoparticles. In these cells a lot of vesicles, probably lysosomes, are already visible. So without nanoparticles these cells
show a somewhat altered ultrastructure compared to the earlier results. Therefore the other two concentrations are compared to these control cells. Not all the results are given in this figure. Compared to these control
cells especially the cells exposed to 0.1 mg/ml nanoparticles seem more affected. In the picture 30 minutes of
0.1 mg/ml the cell has a different shape than normal. Other cells exposed to these concentration are affected
but not always as bad as the cell shown in figure 5 after 30 minutes. After 2 hours exposure to 0.1 mg/ml the
cells are affected badly and a lot of nanoparticles and cell debris were found around the cells. The cells exposed to 0.01 mg/ml nanoparticles are affected only a little. The cells contain more and larger vesicles compared to the control and nanoparticles can be found accumulated inside the cells.
In figure 9 the effects of the nanoparticles during the three weeks studied with EM were shown. Again the
control cells are for some reason a little bit affected. Compared to these cells the cells exposed to both concentrations of nanoparticles are more affected. At the highest concentration (second column) a lot of vesicles
are visible inside the cells. After 4 hours the cells are affected a lot. A lot of cells are partly broken down or
completely dead. Nanoparticles can be found everywhere, because in most of the cells the cell structure is
completely gone. During the three weeks all the dead and very affected cells are broken down or lost during
the medium change and the other cells are trying to recover. Vesicles are still visible and the nanoparticles will
be accumulate inside those vesicles. After 3 weeks nanoparticles can still be observed within vesicular structures in some of the cells (figure 9). The cells are arranged in complete cell layers again and in the cells more
structure is visible. For the lowest concentration (0.01 mg/ml) almost the same results were found. After the
cells are exposed to the nanoparticles, the nanoparticles accumulate inside the cells and the cells are affected.
This can be seen as larger vesicles inside the cells. Nanoparticles are not always found inside the vesicles,
sometimes it looks if they lay in the cytoplasm, not surrounded by a membrane. The shape of the cells is also
affected and during the three weeks, recovery of cells and cell layers takes place. At the end cells are positioned in layers again and cell structures seem normal. Still nanoparticles are accumulated inside the cells.
Long repetitive exposure
With light microscopy the long term exposure of cells to the concentration 0.01 mg/ml was studied. The results were compared to the same concentration that was exposed only once. In figure 10 two pictures are
shown of cells exposed to 0.01 mg/ml F380 nanoparticles after 3 weeks. In the left picture the cells are exposed twice a week to the nanoparticles. So these cells are exposed to the nanoparticles for 6 times. In the
cells a lot of vesicles are visible. It is not possible to distinguish in which cell layer the vesicles are visible, but
probably the upper layer. This one is exposed the most to the nanoparticles. In the right picture the cells were
exposed to the nanoparticles only once: 3 weeks before the moment the samples were collected. In these cells
only a single vesicle was visible. Based on the light microscopic study it is possible to conclude that cells exposed for more than once will be affected more by the nanoparticles. Probably every time nanoparticles are
added, cells will react (almost) the same they did the first time. If this is true it’s not possible to determine
based on light microscopic pictures. To study the cells in more detail electron microscopy was needed. Only a
few samples can be studied with EM, so first the viability (number) of the cells was studied with the SRBassay. Only the most interested moments where chosen to prepare for EM.
14
Figure 9: EM pictures of Rat-1 fibroblast cells exposed to different concentrations of F380 nanoparticles the first 3 weeks. In the left
column the control cells are shown. These cells have already some vesicles inside. Compared with the control all the cells exposed with
nanoparticles are more affected. The cells exposed to the highest concentration (2 nd column) show vesicles inside the cells. Cells exposed to the lowest concentration show also vesicles inside the cell, with inside the nanoparticles, but also affected cell structures and
shapes. At the end (after 3 weeks) the cell culture has recovered: More cell layers are visible. Individual cells still have vesicles with
nanoparticles inside. Between the short and long exposure no large differences were found. Only differences is the number of cell
layers between the short and long exposure: The short exposure has more cell layers.
15
Figure 10: Rat -1 Fibroblast cells exposed to 0.01 mg/ml F380 nanoparticles after 3 weeks. The left picture shows cells
exposed twice a week for 3 weeks to the nanoparticles. In the cells a lot of vesicles (little white dots) are visible. In the
right picture the cells were exposed only once to the nanoparticles. In these cells less vesicles are visible.
In figure 8 the results of the SRB-assay is shown. The cells exposed twice a week to the nanoparticles are given
by the green line. The first day the viability of the cells drops, as the viability of other exposed cells do. After
longer periods of time the viability is increasing as if the cells are recovering. At the end of the three weeks the
viability of the cells exposed to a lower concentration twice a week is exactly the same as the viability of the
cells exposed to the higher concentration (blue line vs green line, week 3). But this viability level is higher than
the viability after the first exposure to the nanoparticles. Based on the SRB-assay time point are chosen to
study the samples with electron microscopy. Those moments are: 10 minutes, 30 minutes, 1 hour and 2 hours.
In figure 9 the results of the electron microscopic study are shown. The results of the long exposure to nanoparticles are shown in the right column. In this figure can be seen that the cells didn’t react very extremely to
the nanoparticles. Cells internalize the nanoparticles inside vesicles and more and more vesicles were visible.
Cell structures keep recognizable because nanoparticles are inside the vesicles and where not found randomly
inside the cytoplasm. Compared to the short exposure study no extra damage was found later in the experiment. When cells where exposed for more than once to the lower concentration, they look more organized
compared to short exposure to the highest concentration. The large filled vesicles which are created during
these three weeks were at the end still present. Compared to the short exposure of 0.01 mg/ml these cells
look more organized and seem to handle the nanoparticles better.
Discussion
The main questions addressed in this paper are: By which mechanism are nanoparticles internalized into nonphagocytic cells and what are the effects of nanoparticles on cells just after exposure to the particles and after
long term exposure? What happens with the cells when they are exposed more than once to the nanoparticles? Before those questions can be answered first the ideal concentration and the best type of silica was
determined.
Dose response experiment
Based on the results of the dose-response experiment three conclusions can be drawn: one, nanoparticles are
toxic. Cells are affected by the particles: In most of the treated cells large vesicles are visible surrounded by
areas without cell structures, while in control cells these areas were not found. In some cells, especially at the
higher concentrations, parts of the cell are completely broken down. The toxicity is not the same for the two-
16
types of nanoparticle we tested: F200 induced these morphological changes and cell death at lower concentrations than F380, suggesting that the F200 nanoparticles are more toxic than the F380.
Two, most of the nanoparticles are surrounded by membranes, in structures which look like vesicles. This was
only found in the cells exposed to nanoparticles, so the nanoparticles seem to induce these large vesicles.
Especially at higher concentrations vesicles can be found in every cell. Not all the nanoparticles can be found
inside vesicles. When the cell is dying and when parts of the cells are already broken down, nanoparticles can
be found individually or in clusters inside the cytoplasm in those affected areas. It is difficult to say if the nanoparticles inside the cytoplasm are the reason the cell dies or that the nanoparticles are inside the cytoplasm
because of the dying cell. Also large vesicles without nanoparticles were visible, especially at lower concentrations. Whether these vesicles are in fact completely empty is difficult to say because in this experiment only
samples of 50 nm were used for electron microscopy. The vesicles are larger than 50 nm, so it is possible that
there are nanoparticles inside the vesicles, but they were lost during the preparation of these sections. To
investigate this a tomogram was made of a 400 nanometer thick section (0.0625 mg/ml of F380 nanoparticles). Inside this sample an almost complete vesicle was found with inside a lot of nanoparticles. These nanoparticles reside in the middle of the vesicle so it is possible to cut next to the nanoparticles (figure not given).
Another explanation for these ‘empty’ vesicles could be that these vesicles are created due to the presence of
the nanoparticles as part of the apoptotic process. Because of the nanoparticles inside the cell, some processes in the cell were activated and the cell will die. These processes could activate the formation of those vesicles.
The third conclusion is that the uptake of nanoparticles is concentration dependent because at higher concentration more affected and dead cells are visible. And more nanoparticles can be found in the affected cell.
It is remarkable that two different type of silica nanoparticles, which are almost the same, causes very different effects on the same type of cells. Every type of nanoparticle and every type of cell has its own properties.
This is because there are a lot of factors determining the effects of nanoparticles. The factors can be categorized into three important categories. The first category depends on the chemical composition of the material:
Solubility, degradability, electrostatic binding, type and composition of material, intrinsic chemical toxicity,
coating, number of molecules on the surface and the release of these molecules (ions). The second category
are the stress stimuli: surface area and roughness, size, shape and concentration. The last category depends on
the environment: the distribution and destination of the nanoparticles, the proteins available in the environment (for example LPS), the intracellular environment, the type of cells with the population doubling time &
agglomeration in relevant media 1-3, 5-9, 18-23. Besides when nanoparticles are modified, for example with a coating, properties will also be changed to unique characteristics: high conductivity, strength, durability and chemical reactivity, for example3.
Even in all these experiments a factor was found which could have had influence on the effects of the nanoparticles. When the results of the dose-response experiment were compared to the results of the other experiments, the cells react very differently. For the other experiments the concentration 0.1 mg/ml was chosen
because in the dose response experiment the cells exposed to the concentration 0.125 mg/ml looked very
healthy. They were not visibly affected by the concentration of nanoparticles. But in the other experiments the
same concentration affected the cells a lot. For example, when picture K & L of figure 3 are compared with
figure 9, picture 4 hours and 2 days 0.1 mg/ml the cells look completely different. The cells of the other experiments are more affected than the cells of the dose response. This is almost the same concentration exposed
to the same type of cells in the same environment. The only difference is that the cells are not from the same
passage number. The dose response experiment was done on another moment than the other experiments, so
the cells were a little older than the cells used in the other experiments. The cells used for the other experiments are from the same age, as they were done with the same cell culture. The age differences between both
cells is around two weeks. To be sure the age of the cells is a determining factor more experiments are needed. This is because the cells which are used in these experiments were a little bit affected for some reason.
Even in the control of the EM samples of the experiments many lysosomes can be found, as already mentioned. Some reasons for this could be the medium, the age of the cells, the circumstances in which the cells
were cultured or the fact that these cells cannot be used very long. They are probably mutated and will react
in a different way than normal fibroblast cells does. .
Uptake mechanism:
To determine the uptake mechanism of nanoparticles fluorescence antibodies were used to identify by which
mechanism the nanoparticles are internalized in the cell. The fact that the antibodies did react with the RAT-1
17
fibroblast cells themselves, was the reason the set of experiments was not done. It was very difficult to find
antibodies which won’t react with the rat fibroblast cells themselves. Most antibodies are made inside mice
and apparently mice and rat cells are almost the same with the same proteins: a lot of background was found
in almost every concentration of every antibody. In the experiment the best concentrations and antibodies are
mentioned, but still there was a lot of background. These antibodies are not the ideal antibodies for these
experiments with these cells. For that reason other antibodies need to be found, for example antibodies which
are created by goats or rabbits. Another possibility is to use another cell line, not from rats, for example human fibroblast cells.
A second reason why the final fluorescent experiment wasn’t done is because of the nanoparticles. To discover
by which mechanism the nanoparticles are internalized using fluorescence, it would be the best to also have
fluorescent nanoparticles. Until now only fluorescent nanoparticles of 50 nm and larger exist, not nanoparticles around the 7 nm. In the article of Limbach et al they describe that the size of the nanoparticle determines
the way of internalization. Nanoparticles smaller than 50 nm are internalized by diffusion, while nanoparticles
between 250 and 500 nm are internalized through sedimentation5. Bihan et al found in their experiment that
the nanoparticles of 30 nm and larger are internalized inside liposomes, while nanoparticles smaller than 30
nm stayed outside the lipid bilayer 15. Because the size of the nanoparticles is a very important factor fluorescent nanoparticles around 7 nm are needed to compare the fluorescent experiment with the other experiments done in the present study.
Nevertheless, some conclusions can be drawn with regard to the possible uptake mechanism of nanoparticles
of 7 nm. As already mentioned in the introduction there are different possibilities of mechanisms. Based on
the results of the dose response experiment can be concluded that the nanoparticles are internalized by a
process like endocytosis and end up inside vesicles, which are probably lysosomes and endosomes. So the
main mechanism of internalization is by an endocytotic mechanism. This was also mentioned in more articles 2,
6, 12
. But it is still unclear by which type of endocytosis the nanoparticles are internalized: macropinocytosis,
clathrin-mediated endocytosis, caveolae-mediated endocytosis and the clathrin and caveolae- independent
endocytosis. Based on the article of Conner it is possible to distinguish between two of the four different
mechanisms based on time 12. In the uptake mechanism experiment was found that the first nanoparticles
were visible in the cells after 10 minutes. So the t ½ will be longer. Conner et al described in their article that
the caveolae mediated endocytosis is a slow mechanism (t½>20 min). It takes more than 20 minutes to internalize nanoparticles with this mechanism. Another mechanism whereby the time is known is the clathrin- and
caveolin- independent endocytosis. This is a very fast mechanism (t½< 10 sec). When the results of this experiment were compared to the results that Conner et al found, the mechanism of internalization is probably the
slow mechanism, the caveolae mediated endocytosis. Since not all the t½ of all the mechanisms are known it is
not possible to conclude, based on this result, that the internalization of nanoparticles is done by caveolae.
However, to be sure the caveolae mediated endocytosis is the main mechanism a fluorescence experiment
must be done whereby fluorescent nanoparticles of 7 nm will be internalized inside, for example, human fibroblast cells while the cell will be labeled with an antibody caveolin against.
Long term effects
The long term effects were studied in cells exposed only once to the nanoparticles and in cells exposed twice a
week to the nanoparticles. On this way the effects of the nanoparticles was studied to discover what happens
when cells are exposed more than once to the nanoparticles.
Based on all the results in this experiment cells will react as follows to the nanoparticles: As soon the particles
are inside the cell the viability of the cells drops down, especially when higher concentration of nanoparticles
entered the cell. The cell is affected and showed signs of cellular stress and some of the cells will die. When the
cell dies internalized nanoparticles are released again in the medium, as a large mass of nanoparticles (see
picture two hours and four hours of 0.1 mg/ml nanoparticles of figure 5 and 9). This is done, most of the time
by breaking down a part of the cell. Luciani et al suggested that the release of nanoparticles in macrophage
happens by the process of microvasiculation. By this process nanoparticles are release by the cell in vesicles 14.
For non-phagocytic cells this process was not yet described and based on the results of these experiments it is
difficult to say that non-phagocytic cells release the nanoparticles by a same mechanism. The release of the
nanoparticles happens sometimes in vesicles, but most of the time the nanoparticles are clustered together,
between a lot of cell debris, non-membrane surrounded. It is more likely that the nanoparticles are released by
the breaking down of the cell. Other unaffected cells can internalize these nanoparticles. After a while the
cells, which are not very affected, recover from the nanoparticles. At the end of this experiment these cells still
have nanoparticles inside which are collected inside vesicles. So the cells are still affected by the nanoparticles,
18
but they survive the nanoparticles. This suggest that the cells are accustomed to nanoparticles: the cells do not
show cellular stress when nanoparticles are present inside the cell. It is also possible the cells are modified by
the nanoparticles, so they can survive. But to be sure, clearly further studies are needed
The graph of the results from the viability test (figure 8) shows some strange results. For example, a strange
thing is that after one day cells exposed for once to the lowest concentration (red line), have a lower viability
than the cells exposed twice a week (red line compared to the green line, day 1). On this moment both the
cells are exposed only once to the nanoparticles, because 1 day earlier the experiment started and the first
concentration of nanoparticles was added. It was more logical when both the lines were on the same level. A
possible factor could be that the number of cells was not exactly the same. The viability test was done on the
petridishes from which aclar pieces were already collected. If some aclar pieces were a little bit bigger than
others, less cells stay behind in the petridish. But this cannot explain a difference like this.
At the end of this test the viability of the cells increased a lot. This could be an indication the viability is not yet
completely recovered. For that reason it is better to do the viability test again and for a longer period. Now the
test was done only three weeks due to an infection by all the different circumstances. For this reason a sample
is missing in both the SRB-assay and the EM results of studing the long term effects:one exposure with a low
concentration (Short 0.01 mg/ml).
Hildebrand et al. did also a long term experiment on the effects of nanoparticles to cells. They exposed two
types of cells to three different concentrations of nanoparticles (50, 100 & 250 mg/L) for three days. They also
found that the viability of the cells drops down as soon the nanoparticles are added, probably due to cellular
stress. After 3 days the viability of their cells was for 94% recovered 16. In the present study almost the same
results were found: the viability test of the RAT-1 cells showed also a recovery after three days, but compared
to the control cells, the viability is still low. After a week the control cells and the cells exposed to nanoparticles have almost the same viability. After that the viability of the control cells increased a lot with an unknown
reason. The viability of the long term low concentration and the short term high concentration is after 3 weeks
on the same level. This indicates that even a low constant concentration of nanoparticles have the same effects on the cells than a high concentration. This could indicate that the number of nanoparticles inside the
cells are on the same level, which indicates that there is accumulation inside the cell. So even a very low nontoxic, ‘safe’ concentration might not be that save when people are exposed for a longer time to these concentration. This is also a reason why it is very difficult to determine the effects of these particles. The accumulation makes it not easy to predict long-term consequences of nanoparticles exposure and for safety and health.
Another complicating factor , which is not studied in the present study, is the effect of the body on the concentration of the nanoparticles, in other words: bioavailability. In this experiment only the effects of the nanoparticles to the cells were shown, but will cells be exposed to these concentrations of nanoparticles? As soon
the nanoparticles are inhaled, the clearance systems of the lungs, such as the mucuciliary clearance and lung
phagocytes, will clear most of the particles. Only a part of the particles will be translocated through interstitium to the bloodstream. Within the bloodstream the nanoparticles can reach the whole body and every cell
inside the body24. But it is also possible that they are relocated to the lung 22 and now eliminated by the lung
clearance system, or they are taken up by intestines or kidney, whereby they also are excreted from the body.
So the chance that a nanoparticle can reach one individual cell is very small. If a cell is exposed to a certain
(low) level of nanoparticles, a very high dose of nanoparticles must be inhaled to reach this number of nanoparticles in a specific cell. To be exposed to the same number of nanoparticles inside a cell, a very high dose
of nanoparticles must be inhaled. In normal situations such a high level will probably not be reached, but what
if a person is exposed to the particles during his whole life? Does he inhale the number of nanoparticles which
will cause serious damage to the cells? For this reason it is very difficult to determine the risks of nanoparticles. Thereby it is unpredictable to say which concentration will be toxic to the cells. Too many factors will
influence the effects of the particles, but also the internalization process. What can be concluded from this
experiment is that the nanoparticles are not very healthy for the cell and the body, and even a low concentration of 0,01 mg/ml will cause serious damage to cells. This indicates nanoparticles could be a high risk and
people have to be careful with using the nanoparticles in daily used products.
19
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