Use of confocal microscopy to examine cellular uptake of nanoparticles
in murine lymph node cells
Loué Nassar
Abstract
The purpose of this study was to examine the fate of nanoparticles (NPs) injected into the
footpads of mice. Many labs are currently investigation the use of NPs as possible delivery
mechanisms for immunization. In this study, fluorescent NPs were injected into the hocks of
mice. After 24 hours, cells from the draining lymph node were examined to determine the
presence of NPs using fluorescent confocal microscopy. Cells were also stained with anti-IAb
and anti-CD80 to examine the phenotypic markers on the cells. Results show that NPs could be
detected in the draining lymph node. While most NPs were not associated with cells, some did
appear to be internalized. In these cases, the cells appeared to be phenotypically IA+ but CD80-.
These results suggest that NPs could be used as a potential delivery mechanism for vaccines.
Results also suggest that the NPs did not stimulate a strong inflammatory response because the
cells that internalized the NPs did not express the cell activation marker CD80.
Introduction
A confocal microscope works by having a laser provide the excitation light on the sample. The
laser is reflected off a dichroic mirror, and then is directed at two motor-mounted mirrors. The
mirrors move and direct the laser across the field, exiting different parts of the sample. The
emitted light from the sample is then reflected back off of those same mirrors, and through the
dichroic mirror and is focused on a pinhole. A detector measures light coming through the
pinhole. The small depth of field that this provides allows the microscope to have higher
resolution in both the vertical and horizontal planes than a regular fluorescent microscope [1].
Using confocal microscopy, it is possible to examine the cellular uptake of nanoparticles
by phagocytic cells. The microscope will allow for three-dimensional renderings to be made
from several individual, two-dimensional scans, whereas another type of microscope would not
be able to attain the necessary resolution through the entire field to do so. In this way, we can
examine if the nanoparticles are internalized by a cell. This could indicate whether nanoparticles
might be a potential delivery mechanism for vaccines.
L-tyrosine phosphate (LTP) NPs are produced from an emulsion of oil and water. They
are spherical and smooth and also degrade hydrolytically in seven days [2]. Injected NPs will
travel to the draining lymph nodes where they will be perceived as antigens. Once in the node,
they will be trapped by the network of phagocytic cells, such as macrophages and dendritic cells.
Afferent lymph enters the node at several locations, but slowly permeates the tissue, giving the
dendritic cells time to trap antigen. During infection, the flow out through the efferent lymphatic
vessel is drastically diminished in order to help to trap antigen. Once trapped, they are processed
and presented in association with major histocompatibility (MHC) class II molecules by
paracortical dendritic cells. This results in activation of adaptive immunity. When the flow out
through the efferent lymphatic vessel resumes, the concentration of lymphocytes in the efferent
lymph is much greater [3]. This has implications as a potential vaccine delivery mechanism. If
specific epitopes could be loaded into the nanoparticles, these epitopes could possibly be
delivered to APCs in the lymph nodes and subsequently expressed in association with the MHC
molecules and activate T-cells.
One role of APCs is the internalization and processing of exogenous antigens and
subsequent expression of epitopes of the antigen in association with MHC class II molecules on
the surface of the APC. In mice, IA is a class II MHC molecule found on the surface of antigen
presenting cells such as dendritic cells. To trigger T-cell activation, the first step is for a T-helper
(TH) cell receptor to recognize the processed antigen in association with the IA molecule.
CD80 is displayed on the surface of activated dendritic cells during inflammation and
functions as a second, or co-stimulatory, signal in T-cell activation. Normally, CD80 molecules
are expressed only on APCs during infection. When expressed, they bind to the CD28 receptor
on T cells, sending a second signal into the T cell. This induces activation and proliferation.
When the MHC molecule in association with antigen peptide is bound by the T-cell receptor
(TCR), this generates “Signal 1.” When CD80 is bound by CD28 (found on the T-cell surface),
this sends “Signal 2” into the T-cell, upregulating the activation of the T-cell [3].
The purpose of this project is to use confocal microscopy to examine the fate of
fluorescent NPs in the draining lymph nodes of mice. My hypothesis is that NPs will be found
internalized within cells of the lymph nodes and that these cells will express IAb and CD80.
Materials and Methods
Mice
Female C57BL/6 mice between eight and fifteen weeks of age were used. Animals were treated
in accordance with University IACUC policy.
Preparation of nanoparticles
LTP nanoparticles that were loaded with rhodamine were the kind gift of Dr. Yun from
Biomedical Engineering at the University of Akron. NPs were weighed out and resuspended in
phosphate-buffered saline (PBS) so that 30μL contained 200μg of nanoparticles (NPs). NPs were
resuspended by drawing the NPs up into a 1cc syringe, starting with an 18g needle, and gradually
decreasing the bore size to 27g. Once the particles were suspended, they were loaded into a 1cc
syringe for injection.
Injection of mice
The sample was injected into all four footpads of a mouse. After 24 hours, the popliteal, inguinal,
brachial, and axillary lymph nodes were removed.
Preparing cell suspensions
A single cell suspension was prepared. Using forceps, fat tissue was removed from the lymph
nodes. They were then placed on a 100-mesh screen in Hank’s buffered salt solution (HBSS) in a
35x10mm petri dish. The barrel of a 1 cc syringe was used to push the nodes through the mesh in
order to release the cells into the solution. Using a syringe, the cell-containing solution was then
transferred into a 12x75mm test tube. The sample was then centrifuged at 1400 RPM for seven
minutes. After centrifugation, the media was decanted and the cells were washed twice with 2mL
of Ca2+-free, Mg2+-free buffer solution. It was mixed on a vortex mixer prior to each wash. Cells
were then counted and the solution was divided up into four test tubes containing approximately
1.25 million cells per tube.
Cell staining
Each tube was once again centrifuged for seven minutes at 1400 RPM. The solution was poured
off, and the cell pellet was resuspended using a vortex mixer. To one tube, 25μL of PBS was
added. To each of the other three tubes, 25μL of a solution containing antibody to Fcγ III/II
Receptor was added in order to prevent false positives resulting from nonspecific Fc binding.
100μg of this reagent was obtained from BD Pharmingen. For this solution, there was one
microgram of antibody for every 25μL of solution. The tubes were then incubated on ice for 5
minutes. After incubation, the tube containing PBS received another 100μL of PBS, and the
other three tubes received 50μL of a solution containing antibody to IAb conjugated to
fluorescein isothiocyanate (FITC) at a concentration of 100ng/50μL and 50μL of antibody to
CD80 conjugated to Texas Red at a concentration of 50ng/50μL. Both of these antibody reagents
were obtained from BD Pharmingen. The tubes were then incubated on ice for 30 minutes. After
incubation, 1mL of a solution containing HBSS with sodium azide was added to each tube,
followed by centrifugation. Two washes were performed using 1mL of HBSS with sodium azide.
Slide preparation
The cells were placed on clean slides with a drop of a solution of glycerol mixed with a 20mM
solution of sodium azide in HBSS at a 1:1 ratio.
Confocal microscopy
The slides were examined using the Zeiss LSM 510 Meta Confocal Microscope. The fluorescent
excitation wavelengths were set to 485nm and 543nm. Z-stacks were performed with a layer
interval of 0.5μm.
Results
Figure 1 shows that NPs were present in the draining lymph nodes 24 hours post injection. NPs
could be seen to fluoresce at both excitation wavelengths of 485nm and 543nm. Figure 1a shows
that, when observed using both excitation wavelengths, the NP appears yellowish in color.
Figure 2a shows a cell that is positive for IAb and CD80 and is also associating with a NP. Figure
2b shows a cell that is only IAb positive.
a
b
c
Figure 1: Nanoparticle found in the lymph node. (a) Nanoparticle observed with 63x objective under
excitation wavelengths of both 485nm and 543nm. (b) Isolated for excitation wavelength of 485nm.
(c) Isolated for 543nm.
a
b
Figure 2: Lymph node cells stain
positive for CD80 and IA. (a) Scan of
field containing CD80+ cells under
the 63x objective. The cell is
positive for CD80 and MHC class II
and is associated with a NP. (b) A
cell that is only MHC Class II+
viewed with the 100x objective.
It also appears that nanoparticles may actually be taken up by cells in the draining lymph
node. Figure 3 shows the confocal Z-stack of one cell. In this cell, a green layer was visible
surrounding what appears to be red staining material in the cytoplasm, indicating the surface
MHC Class II molecules, IAb, with rhodamine stained particles inside of them (Figure 3). Based
upon the appearance of the red particle only in sections closer to the center of the cell, it would
appear to be a nanoparticle inside the cell as opposed to CD80, which would be found on the
cell’s surface. The fluorescence appeared a bit more diffuse, which may indicate that the
nanoparticle was being processed or broken down by the cell.
Figure 3 An IA+ cell with what appears to be an intracellular NP. (a) The uppermost section of the ZStack showing the beginnings of the IA+ outer membrane. (b) 1.5 μm from top of cell. (c) 2.0 μm from
top. (d) thru (o) consecutive sections beginning at 3.5 μm from top of cell and ending at 9.0 μm into
cell at 0.5 μm intervals. These sections show the appearance and disappearance of what appears to
be a nanoparticle. (p) 10.5 μm into cell. (q) 11.0 μm into cell. (r) The bottommost section of the ZStack at 12.5 μm from the top. All viewed with 63x objective.
Discussion
The purpose of this project was to determine if fluorescent NPs could be detected in the draining
lymph nodes of mice after injections into the footpads. Previous studies have shown in vitro that
NPs are capable of being internalized by and altering function of phagocytic microglia cells in
rats [4]. Another study has shown in vitro that, NPs can be internalized by KB cells and can also
be a vector for nonviral gene delivery [5]. NPs have also been shown in vivo to have potential as
nonviral vectors for gene delivery into the mouse ventral midrain and lateral ventricle [6]. LTP
NPs such as those used in this research have also been shown to be internalized by human
fibroblasts and show controlled transfection and can be used as a non-viral gene delivery vector
[2,7]. This study provides evidence of in vivo NP internalization in the lymph node. These
particles were provided Dr. Yun from Biomedical Engineering at the University of Akron.
Here we show that NPs can be seen in the draining lymph node. Because there is so much
rhodamine loading into the NPs, their emission wavelengths bleed over into the emission
spectrum of the FITC. This is probably because the emission spectrum of rhodamine, while it
peaks in the red wavelengths (around 700nm), some of its emission spectrum does fall in the
550nm region that produces green fluorescence. I believe this is why they appear yellow when
viewed under both excitation wavelengths.
Using the ability of the confocal microscope to provide three-dimensional images, it
appears that NPs can be seen in cells the lymph node. It is important to note that the cells
internalizing the NPs expressed only MHC class II, and not CD80. Because CD80 is only
expressed on the surface of activated dendritic cells, this indicates that the NPs themselves are
not immunogenic and do not activate APCs. It is possible that, when loaded with an antigen
peptide, NPs might be more immunogenic because they might be more likely to stimulate
internal toll-like receptors (TLRs). We could also manipulate the NP surface to target specific
cell-surface TLRs to activate an even more specific immune response.
In conclusion, the presence of a nanoparticle inside a cell, especially a particle that was
being processed, provides evidence that nanoparticles may indeed be a possible delivery
mechanism for immunization against certain types of antigens. If a nanoparticle could be loaded
with a known epitope for a specific antigen, it is possible that a more targeted response could be
induced.
This also has implications for the use of confocal microscopy. Confocal microscopy
could be used to further determine whether or not nanoparticles really do associate with cells of
the immune system, and to what extent they associate. The higher contrast and higher resolution
images that it can provide can allow for more conclusive imagery and imaging of living, still
functional cells. It could be possible to try to follow the procedure again, but record what
happens to the cells and nanoparticles over time. A cell that takes up a nanoparticle could be
observed to determine a more exact process.
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