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Targeting of human dendritic cells with surfaceoptimized Nanoparticles
by
Stefan Hellberg
Thesis
submitted to
University of Appllied Sciences
Bonn-Rhein-Sieg
Department of
Natural sciences
for the degree of
Bachelor of Science in Applied Biology
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Abstract
The medical importance of Nanoparicles has strongly increased over the recent
years. In terms of cancer research they offer great opportunities as a drug delivery
system. Our group performs basic research in the field of Graft vs. Host disease
(GvHD). For the better understanding of the role of Dendritic cells (DCs) in GvHD
development a series of investigations was started lately, with the aim of efficiently
labeling Dendritic cells (DCs) with surface modified Nanoparticles loaded with two
different fluorescent markers. The focus of this paper was the evaluation of four
small styrene based polymeric nanoparticles ranging from 125 to 153 nm in
diameter with narrow size distribution. In an in vitro approach we showed that all
particles were readily taken up at concentrations ranging from 25 g/ml up to 300
g/ml while showing little to no toxicity in a flowcytometric measurement. A
fluorescent microscopy analysis confirmed the intracellular location of the particles.
In an additional in vivo study we monitored the distribution of the particles during a
period of 96h after intravenous administration and demonstrated the presence of
particle in liver, spleen lungs and skin. This work revealed important differences in
the properties of the particles regarding the tested aspects that influence their
potential for medical application.
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Table of contents
1. Introduction ..................................................................................................... 1
1.1 The role of Dendritic cells in the immune system ...................................... 1
1.2 Dendritic cells, role in the development of an acute Graft vs. Host disease
(GvHD)................................................................................................................ 21.3 Nanoparticles, medical and pharmacological relevance .............................. 3
2. Materials and Methods .................................................................................... 4
2.1 Materials .................................................................................................... 4
2.1.1 Equipment .......................................................................................... 4
2.1.2 Chemicals and consumption objects .................................................. 5
2.1.3 Surgical instruments ........................................................................... 6
2.1.4 Drugs, Media and Additives ................................................................ 7
2.1.5 Zytokines ............................................................................................ 72.1.6 Cellculture media ................................................................................ 7
2.1.7 Buffer and Solutions ........................................................................... 8
2.1.8 Antibodies ........................................................................................... 8
2.1.9 Laboratory animals ............................................................................. 9
2.1.10 NSG Mouse haltung/ftterung ......................................................... 9
2.1.11 Nanoparticles .................................................................................. 9
2.2 Methods .................................................................................................. 10
2.2.1 Isolation of peripheral blood lymphocytes ......................................... 10
2.2.2 Generation of dendritic cells (DCs) from blood monocytes ............... 10
2.2.3 Cryo-conservation of cells ................................................................. 11
2.2.4 Flow cytometry ................................................................................... 11
2.2.5 Confocal laser scanning microscopy ................................................ 13
2.2.6 In vitro examination of DCs with NP ................................................. 13
2.2.7 Extraction of organs / preparation of in vivo samples ....................... 14
2.2.8 Biofluorecence Imaging (BFI) ........................................................... 14
3. Nomenclature, Abbreviations and Units ........................................................ 15
4. Results .......................................................................................................... 15
4.1 In Vitro Examination of DCs loaded with NP ........................................... 15
4.1.1 Viability determination of DCs ........................................................... 16
4.1.2 Determination of NP uptake into DCs ............................................... 20
4.1.3 cLSM results for NP GB-PS 62 ......................................................... 21
4.1.4 Results NP 61 ................................................................................... 24
4.1.5 Results NP 63 ................................................................................... 27
4.1.6 Results NP 59 ................................................................................... 28
4.2 Biodistribution of NP in a Mouse model ................................................... 32
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4.2.1 Mouse kinetics .................................................................................. 32
4.2.2 Organ distribution ............................................................................. 39
5. Discussion ..................................................................................................... 42
6. References .................................................................................................... 47
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1. Introduction
1.1 The role of Dendrit ic cel ls in the imm une sys tem
When foreign microorganisms enter the body, usually the host system is able to
detect them immediately when they enter the peripheral tissue and trigger first line
defense mechanisms in an unspecific response known as innate immunity. During
evolution this mechanism of protection has evolved as a very effective and fast
way of dealing with the vast majority of the encountering contagious material. The
cells of the innate immune system do recognize antigen of common
microorganisms and are able to control an infection. However, some pathogens
have developed strategies to avoid this front line detection and cannot be detected
by the usual means of the innate immune response. In this case a more versatile
mechanism of defense is provided by the lymphocytes of the adaptive immune
system which also offer an increased protection against subsequent reinfections.
Like macrophages, granulocytes and monocytes DCs are part of the myeloid
lineage which is derived from a common myeloid progenitor. [1]
DCs play a crucial role in the initiation of the adaptive immune response. In their
immature state DCs migrate through the blood to the tissues where they reside
and survey their environment. They constantly ingest large amounts of the
surrounding extracellular fluid via macropinocytosis and are capable of receptor
mediated phagocytosis if they encounter common features of pathogens like
bacterial cell wall proteoglycans. Once they have ingested antigen, DCs stop all
phagocytic and macropinocytic activity. Their maturation begins and they migrate
through the afferent lymph to a regional lymph node, where they begin to recruit
nave (antigen inexperienced) lymphocytes. Mature DCs (mDCs) are highly
effective antigen presenting cells (APCs) with the ability to activate pathogen
specific lymphocytes that they encounter in the lymph nodes, a process is known
as priming. Although almost every type of cell hast the ability to present antigen,
the term APCs only refers to the so called professional antigen presenting cells
like DCs, macrophages, monocytes and B-lymphocytes. [2,3]
Hence DCs function as a mediator between cellular and adaptive immune system.
Immature DCs act as sentinels with the ability to ingest and process antigen, which
only have a weak ability of stimulating T-cells. Immature DCs bear different lectin
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receptors which serve as antigen receptors and are also involved in the regulation
of migration and interaction with lymphocytes. The maturation is stimulated by the
uptake of bacterial or inflammatory substances. Upon maturation the DCs start to
present the ingested antigen on the surface through major histocompatibility
complex class II (MHC-class-II) receptors. Beneath the presentation of antigen, achange in the morphology and in the expression of co-stimulatory receptors like
CD80 (Cluster of Differentiation 80) and CD86 is part of the maturation process
and makes it possible to generate a T-cell response. The presented antigen is
recognized by the T-cell receptor (TCR) of T-lymphocytes, which stimulates their
clonal proliferation. [4]
1.2 Dendrit ic cel ls, role in the development of an acute Graft vs.
Hos t disease (GvHD)
Dendritic cells (DCs) can be activated during the conditioning therapy before an
allogeneic hematopoietic stem cell transplantation (HSCT) caused by tissue
damage. DCs are antigen presenting cells (APCs) which, once activated, have the
ability to stimulate T-Cells. This can lead to complications after an HSCT as the
DCs can activate the donor T-lymphocytes which can lead to an acute GvHD, as
the donor T-cells start to react against the recipient tissue. [1] The occurrence of
GvHD could be avoided by absence of DCs in the recipient as shown in a mouse
model. [5]
There are still open questions in the role of DCs in GvHD development. It is still
unclear if stimulation of recipient T-cells is triggered in the secondary lymphatic
organs or in the end organ. Also DCs can potentially constrain the T-cell reaction
through regulatory mechanisms like the Programmed Death (PD) ligand-1 and its
receptor PD-1 on T-cells. They may even completely avoid a T-cell answer in an
inactivated state. [6]
Potential therapies like the systemic application of DC depleting agents are a
promising approach. The usage of Nanoparticles to transport either depleting or
manipulating substances into the cells can be advantageous. They enable the
transport of larger amounts of substance than single molecules do. At the same
time they offer the opportunity to target specific cell types like DCs. Current in vitro
studies of antibodies against the differentiation marker CMRF-44 and against the
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activation marker CD83 are already available [7]. Although for the targeting of
recipient DCs with antibody an adequate preclinical in vivo model is yet to be
found.
1.3 Nanoparticles, medical and pharmacologicalrelevance
During the last years many the development of nanotechnology has made great
progress and new forms and structures of nano materials have become available.
Nanotechnology has made its way into many facets of our lives. New materials
with extraordinary properties have been developed. The probably most famous is
the lotus effect, but there are many structures of interest. Fullerenes like carbon
nanotubes are very interesting because of mechanical strength, electrical andchemical properties. Also nanoparticles are especially of interest especially for
their chemical, optical, magnetic and electrical attributes.
In the field of medicine and pharmacology nanoparticles have shown to be
advantageous because of their high loading efficiency due to their capacity and
their very high mass/surface ratio in contrast to the pure active substances often
aggregates when pulverized. Nanoparticles can also preserve the original
substances until they reach their target [8]
Hydrophobic agents for example can be loaded into particles, creating a
hydrophilic surface enabling a pharmacological application. [9] Nanoparticles can
be used to surpass biological barriers like the blood-brain barrier and they possess
the ability to be taken up by cells via endocytosis.
Also the surface of nanoparticles can be modified with a great variety without
affecting the cargo. Many of the current studies are researching the cellular uptake
depending on functionalization of the surface, size and shape of the particles [10]
In our studies we surveyed the uptake and toxicity of different particles supplied by
the MPIP into dendritic cells in an in vitro model. The particles were created via a
mini emulsion process. All particles are of similar size ranging from 120 nm and
160 nm in diameter. The main difference exists in their functional groups, two of
the particles are not functionalized, one particle is amino functionalized and one
particle is Carboxyl functionalized. The particles are triple loaded with two different
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fluorescent dyes, BODIPY and IR-dye-780, and with Platinum(II)acac, an
additional contrast agent. In our in vitro model we cultivated DCs and loaded them
with NP. The cells were analyzed via fluorescence activated cell sorting and
confocal laser scanning microscopy. An additional study was performed in an in
vivo approach in which the different particles were injected intravenously intoNOD.Cg-Prkdcscid Il2rgtm1wjl mice to survey the particle bio-distribution. During a
period of 96h the animals were monitored via biofluorecent imaging (BFI). The
animals were then sacrificed and the distribution of NP to the organs was
investigated.
Upcoming experiments with the aim of monitoring of Nanoparticle loaded DCs
after intravenous administration will allow further conclusions on the function of
DCs and their migration. Another important step will be the introduction of a
humanized mouse model in order to investigate potential medical fields of
application.
2. Materials and Methods
2.1 Materials
2.1.1 Equipment
Air liquide Espace 331 Tec Lab (Knigstein)
Autoclave KSG Sterilizers (Olching)
Centrifuge Kendro-Heraeus(Langenselbold)
CO2-Incubator 37C, 5% CO Heraeus (Langenselbold)
Confocal laser scanning microscope (CLSM) 510-UV
Zeiss (Oberkochen)
Flow cytometer Canto with Diva software BD Biosciences (Heidelberg)
Freezer -80C Heraeus/Kendro(Langenselbold)
Fume hood bench type Fume Adsorber TAZ 19 Medite (Burgdorf)
Ice machine UBE50/35 Ziegra (Isernhagen)
In Vivo Imaging System (IVIS) Xenogen
Microliter pipettes transfer pipette R S0,5-10l / 10-100k / 20-200l / 100-1000l
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Nitrogen-Cryo-bank XLC 1370, MVE Europe (Solingen)
Nitrogen tank Taylor-Wharton XL-180 Tec Lab (Knigstein)
Phase contrast microscope for cell culture Axiovert 25, Zeiss (Jena)
Pipette aid PipetBoy IBS Integra Biosciences(VWR Darmstadt)
Refrigerator and freezer combination4C / -20C
Privileg (Frth)
Vortex MS2 Minishaker IKA (Staufen)
2.1.2 Chemicals and consumption objects
7-Aminoactniomycine BD Bioscience
(Erembodegem, B)Beaker (glass) 200,500 and 1000 ml Schott (Mainz)
Cell strainer BD Falcon
Cell culture flask250 ml culture flask
Greiner (Nrtingen)
Counting chamberFuchs-Rosenthal
Schreck (Hofheim)
Cover glasses 24x32 mm Menzel (Braunschweig)
Culture plates24-, 48-, 96-well Culture plates
Greiner (Nrtingen)
Disposable pipettes, sterile2, 5, 10, 25 and 50 ml
Greiner (Nrtingen)
DMSO (Dimethyl sulfoxide) Merck (Darmstadt)
DRAQ5 Biostatus (Shepshed, UK)
EDTA (Ethylenediaminetetraacetic acid) Sigma (Deisenhofen)
Eppendorf tubes 200 l, 500 l, 1500 l Eppendorf (Hamburg)
FICOLL-paque GE Healthcare (Freiburg)
Freezing-boxes Nalge Nunc (Wiesbaden)
Freezing-tubes Cryotube 1.8 ml Nunc (Wiesbaden)
Freezing-tube rack Roth (Karlsruhe)
FACS-tubes for Flow cytometryBD Falcon
BD Bioscience(Erembodegem, B)
FACS-tube rack Roth (Karlsruhe)
Falcon tubes50ml BD Falcon tubes
BD Bioscience(Erembodegem, B)
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Falcon tube racks BD Bioscience(Erembodegem, B)
Gloves (latex) Semperid (Austria)
Insulin syringe0,3 ml 30 G 8mm
Becton Dickinson(Heidelberg)
Isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane)
Abbott (Wiesbaden)
Paraformaldehyde (PFA) Merck (Darmstadt)
PBS (Phosphate-buffered saline), liquid Gibco BRL (Karlsruhe)
Petri-dishes 94mm, 25mm Greiner (Nurtingen)
Pipette tips0,5-10 l , 10-200 l, 100-1000 l
Starlab (Ahrensburg)
Sodium chloride (NaCl) Carl Roth (Karlsruhe)
Syringes2 ml single-use syringes
Braun (Melsungen)
Trypan blue Merck (Darmstadt)
2.1.3 Surgical instruments
Clamps
Straight Clamp 12.5 cmCat. Nr. 14009 - 12
Forceps
Straight forceps 10 cmCat. Nr. 11050 - 10
Forceps with curved with slotted 0.8 mm x 0.7 mm tip, 10 cmCat Nr. 11052 - 10
Straight forceps 12 cm
Cat. Nr. 11002 - 12
Scissors
Surgical Scissors straight sharp/blunt blade 12 cmCat. Nr. 14001 - 12
Surgical Scissors straight sharpCat. Nr. 14002 - 12
All Referring to FST - Fine Science Tools (Heidelberg)
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2.1.4 Drugs, Media and Additives
Aqua dest. B.Braun (Melsungen)
AIM-V Medium Gibco BRL (Karlsruhe)
Ketamin (50 mg / ml) Ratiopharm (Ulm)
Heparin (Liquemin) Roche (Grenzach-Wyhlen)
Human albumin Octapharm (Langenfeld)
Human Serum (HS) from healthy donor blood,heat-inactivated at 50C for 30 min. Mixed from 10to 20 donors. .
Blood bank of Universityhospital (Mainz)
Penicillin/Streptomycin Gibco BRL (Karlsruhe)
2.1.5 Zytokines
Interleukin 1 (IL-1) Miltenyi (Bergisch-Gladbach)
Interleukin 4 (IL-4) R&D Systems (Wiesbaden)
Interleukin 6 (IL-6) Miltenyi (Bergisch-Gladbach)
Granulocyte macrophage colony-stimulating factor(GM-CSF)
Bayer (Leverkusen)
Tumor necrosis factor (TNF- ) Promokine (Heidelberg)
Prostaglandin E2 (PGE2) Sigma (Deisenhofen)
2.1.6 Cellculture media
DC-Medium AIM-V with 1% humanserum
Medium A DC-Medium with GM-CSF
800 U/ml and IL-4 1000 U/mlMedium B DC-Medium with GM-CSF
1600 U/ml and IL-4 1000U/ml
Medium C DC-Medium + with GM-CSF800 U/ml and IL-4 500 U/ml
Freezing medium AIM-V with Humanalbumin 8% and Heparin(Liquemin) 10 U/ml
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2.1.7 Buffer and Solutions
FACS buffer
storage at 4C
PBS0.5% BSA
500ml250mg
Narcosis solution
storage at 4C max. 14 days
Xylazin (Rompun 2%)Ketamin (50 mg/ml)H2O
0.8 ml2.0 ml2.8 ml
10 mg/kg body weight Xylazin and 62.5 mg/kg body weight Ketamin were applied.
This equals 80 l per animal based on an average weight of 30 g.
Trypan blue (stock solution) Trypan blueH2O
2,0 gad 1 l
Trypan blue (application solution)
storage at RT
Trypan blue stock solution150 mM NaCl
75 ml25 ml
2.1.8 Antibodies
Antibodies used for flow cytometry as following
Labeling Specificity Conjugation Producer
CD45 Mouse FITC / PE / APC Beckman Coulter
CD11c Mouse APC Beckman Coulter
CD80 Mouse FITC Beckman Coulter
CD83 Mouse FITC Beckman Coulter
CD86 Mouse PE Beckman Coulter
CD14 Mouse PE Beckman Coulter
CD19 Mouse FITC Beckman Coulter
CD3 Mouse APC Beckman Coulter
HLA-DR Mouse PE Beckman Coulter
Tab.1: Overview of antibodies used to label surface antigens. The followingconjugations were used: Fluoresceinisothiocyanate (FITC), Phycoerythine (PE)and Allophycocyanine (APC)
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2.1.9 Laboratory animals
In our invivo studies NOD.Cg-Prkdcscid Il2rgtm1wjl mice also known as NOD scid
gamma mice were used. The animals were supplied by the Jackson laboratory in
bar harbor (MA, USA; stock number 005557). The following acceptance
application was existent: "In vivo Markierung menschlicher dendritischer Zellen
durch optimierte Nanopartikel im humanisierten Mausmodell", file number 23 177-
07/G 11-1-014
2.1.10 NSG Mouse haltung/ftterung
Stock breeding and animal manipulations were performed by the central laboratory
animal facility (ZVTE, Zentrale Versuchstiereinrichtung) of the Johannes
Gutenberg university medicine under specific pathogen free (SPF) conditions.
Drinking water supply was supplemented with 0.08 mg/ml Borgal (Sulfadoxinum,
Trimethoprinum) after sterilization by autoclavation.
2.1.11 Nanoparticles
For all experiments conducted Polystyrol based polymeric Nanoparticles (NP)
were triple loaded with IR-dye-780, BODIPY and Platin(II)acetylacetone
(Platin(II)acac). The particles vary in size, functional groups, and surfactant used.
The following Nanoparticles were provided by the Max-Planck-Institue for polymer
research (MPIP)
ParticleFunctional
groupSurfactant
Diameter, nm(STDN, %)
GB-PS-61(NP 61)
- Lutensol AT 50 145 (17.7)
GB-PS-62
(NP 62)COOH Lutensol AT 50 139 (14.0)
GB-PS-63(NP 63)
NH2 Lutensol AT 50 153 (8.9)
BR 59(NP 59)
- SDS 125 (15.7)
Tab.2: Overview of Nanoparticles used in the experiments. Concentration of NPsamples is 3.7% and were provided by the . All NP are triple loaded with a) IR-dye-780 b) BODIPY c) Platin(II)acac
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2.2 Methods
2.2.1 Isolation of peripheral blood lymphocytes
Peripheral Blood Mononuclear Cells [PBMCs] were isolated from healthy human
donor buffy coat products via density gradient separation. Donor blood wasobtained under consideration of the declaration of Helsinki. One healthy patient
buffy coat is derived from approximately 500ml of peripheral blood
FICOLL was used as separation medium to create a density gradient. 15 ml
FICOLL were added to a Falcon tube and centrifuged shortly until FICOLL is below
the frit. At least 15 ml buffy coat was added to the tube and the remainder was
filled up with PBS. The sample was centrifuged for 20 minutes at 2300 rpm
without break and without acceleration. The PBMC band should now be visible aswhite ring between FICOLL and blood plasma and can be transferred into a new
falcon tube without a frit. The sample was washed three times with PBS.
If more two or more tubes have been used they were now pooled in one tube. The
tube was filled up with PBS and the total number of cells was estimated using a
Fuchs-Rosenthal counting chamber and trypan blue as a dye. About 500 Million
PBMCs can be derived from one buffy coat.
2.2.2 Generation of dendritic cells (DCs) from blood monocytes
Blood monocytes are isolated from PBMCs via plastic adhesion using standard six
well plates. 15 Million PBMCs in 3ml DC-Medium (AIM-V with 1% human serum if
no other composition is indicated) are seeded into each well. After one hour of
incubation non-adherent cells are removed with the supernatant and washing
three times with PBS. The cells are incubated in 3 ml fresh Meduim A to stimulate
maturation into dendritic cells (DCs). After 48h 800l medium is removed from
each well and pooled into a Falcon tube. After 5 minutes of centrifugation at 1500
rpm the supernatant is discarded. The cell pellet is resuspended in 1 ml Medium B
and distributed equally to the wells. After another 48h of incubation this procedure
is repeated, followed by another 24h of incubation. After a total of 6 days the
generation of immature dendritic cells is completed. The supernatant is collected
and the remaining cells are harvested by incubation with cold PBS/EDTA followed
by rinsing the wells several times with cold PBS until most of the cells have
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detached from the surface and pooled together with the supernatant. The total cell
count is estimated and the cells are ready for further processing.
2.2.3 Cryo-conservation of cells
To preserve cells for longer periods of time, the cells were stored in a Nitrogen
tank. To prepare the cells for cryo-conservation the cells were centrifuged down at
1500 rpm for 5 min and re-suspended in freezing medium containing 10% DMSO.
The desired volume and the resulting cell concentration was depending on the
further usage. After the addition of the DMSO containing medium the cells were
distributed into the according number of cryo tubes with 1 ml per tube and
gathered in Isopropanol containing cryo-conservation boxes as quick as possible.
After pre-cooling in a -80C fridge the tubes were transferred into the Nitrogenbank for long time storage at -196C.
DCs were stored at a concentration of 1.0-2.0 x 106 cells/ml and PBMCs were
stored at a concentration of 2.5-5.0 x 107
2.2.4 Flow cytometry
Beneath size and granularity of a cell the expression of different surface molecules
can be used to distinguish between different populations of cells. In flow cytometry
this selective expression can be detected via FACS (fluorescence activated cell
sorting) analysis. For this purpose antibody specific to the cellular structures of
interest is coupled to a fluorescent dye (FITC, PE, APC). The fluorescence of each
dye is triggered by the excitation with light of a specific wavelength. The
fluorochromes respond with the emittance of light (usually) at a different
wavelength. The wavelength at which the maximum excitation is reached and the
wavelength at which the emission shows a peak are unique properties of afluorochrome. For example APC is excited and emits in the red spectrum of UV
light. The maximum excitation is maintained at a wavelength of 650 nm, the
emission peak for APC is at 660 nm. In comparison FITC has an excitation
maximum at 494 nm while the emission peaks at 520 nm which lies in the range of
green UV light.
This permits to distinguish between the different dyes. Hence the application of
three different fluorochromes makes it possible to analyze the expression of up to
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three different surface marker proteins of a cell at the same time.
For the actual measurement, the sample solution is routed through a laminar flow
where each cell or particle (event) passes a laser beam individually. This
stimulates the fluorescence of a conjugated dye which leads to an emission signal
at the according wavelength. The emission intensity is detected and recorded for
any event and can be assigned to the corresponding dye in the analysis later on.
Additionally the size and granularity is determined for each event. Light that is
scattered by the particle in a forward direction relative to the axis of the incident
light is recorded through the forward scatter (FSC). The intensity of the FSC signal
is proportional to the size of a particle. The side scatter (SSC) collects the light that
is deflected to the side at a 90 angle relative to the incident light. The SSC is
proportional to the granularity of the according event.
Usually an amount of 104 viable cells are analyzed in a flow cytometer per sample.
For each sample an amount of 1 x 105 up to 2.5 x 106 are needed. The required
amount was centrifuged down, re-suspended in FACS buffer and distributed to
FACS tubes. After another centrifugation step the supernatant was discarded and
2.5 l of a directly fluorochrome-coupled antibody was added. The samples were
incubated for 15 min at 4C. Subsequently excess antibody was washed off with
FACS buffer. Finally the cells were fixed in 500 l PBS containing 1 % of
Paraformaldehyde (PFA) allowing them to be stored up to one week at 4C before
measurement.
To additionally perform a cell viability confirmation the cell samples can be stained
with fluorescent dyes like 7-Aminoactinomycine (7-AAD) or Propidiumiodide (PI).
In our experiment we used 7-AAD allowing us to discriminate between viable,
apoptotic and dead cells.
The 7-AAD staining was performed after the normal staining with FACS antibodies
instead of the fixation step. As the cells were not fixated the could only be stored
for up to three hours before they were analyzed. The sample tubes were
centrifuged down and the supernatant was discarded. 300 l PBS and 20 l 7-
AAD solution (0,2 mg/ml) were added to each tube. After 15 min. incubation at 4C
650 l PBS were added. The tubes were centrifuged and the supernatant was
discarded to wash off excess 7-AAD. The cell pellet was re-suspended in 300 l
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PBS and stored at 4C in darkness until the measurement was performed.
[2] http://www.bdbiosciences.com/research/multicolor/spectrumguide/index.jsp
2.2.5 Confocal laser scanning microscopy
During confocal laser scanning microscopy (confocal laser scanning microscope,
cLSM) fluorescent dyes are activated via irradiation with a laser at a specific
wavelength. The emission of the fluorescent dye can be detected, allowing
conclusions concerning the locations of the fluorescent dye. For this purpose the
laser beam is directed through a raster unit onto the object of interest. The
resulting emission is detected by a photo multiplier unit.
The confocal mode of the cLSM enables the scanning of cells plane by plane while
interference signals are from other planes are suppressed as the excitation- and
detection focus are directly on top of each other. This is of special interest as it
allows studying the interior of a cell. At the same time cLSM has the advantage
that living cells can be investigated. This gave us the opportunity to detect the
intracellular particles by the fluorescent emission of the particles in living DCs. To
permit particle detection we stimulated the fluorescence of the incorporated
fluorescent dye BODIPY (excitation / emission maxima ~503 / 512 nm). The
plasma membrane of the cells was stained with CellMask Orange (556 / 572
nm). Nuclear staining was performed using HOECHST 33342 cell-permeable DNA
stain (346 / 497 nm) except for one experiment where DRAQ5 (646 / 681 nm) was
used instead.
2.2.6 In v i t roexamination of DCs with NP
iDCs were incubated with NP to stimulate NP uptake during the maturation
process For this purpose iDCs were seeded into 48-well plates, 150K DCs in 1 ml
DC-Medium per well. 1-2 h of incubation at 37C are needed to let the cells
adhere and recover. Then the NP was added to the cells in different concentrations.
(The concentrations 25 g/ml; 75 g/ml ; 150 g/ml ; 300 g/ml as well as negative
controls without NP were applied).
The cells were incubated overnight at 37C.The supernatant of each well was
discarded. To get rid of excess NP the wells were rinsed one time with DC medium.
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If too many cells had detached another 1-2 h of incubation were needed for the
cells to settle. Otherwise, the DC medium was discarded and the cells were
supplemented with 1 ml Medium C per well plus an additional cytokine mix (IL-1
[10 ng/ml], TNF- [10 ng/ml], IL-6 [1000 U/ml] and PGE2 [1 g/ml]) to stimulate DC
maturation. After 48h of incubation at 37C the mature DCs (mDCs) wereharvested with cold PBS (4C) and prepared for further analysis.
2.2.7 Extraction of organs / preparation of in vivo samples
To gather the required samples all animals were narcotized with narcosis solution
(10 mg/kg body weight Xylazin and 62.5 mg/kg body weight Ketamin). A blood
sample was obtained via heart puncture with a 2 ml syringe and a 30 mm needle
through the abdomen The extraction of organs was performed after previouscervical dislocation. With aid of scissors and forceps a skin sample was taken from
the back of the mouse (about 1 cm2). After opening the abdominal wall by cutting
through the coat, spleen, liver and lungs were removed. All samples were stored
on ice in PBS and analyzed immediately.
2.2.8 Biofluorecence Imaging (BFI)
Different concentrations of NP diluted in PBS through were injected intravenouslyinto mice of different ages. The localization of the particles was surveyed over a
period 96h after injection (pictures are taken after 0h, 4h, 6h, 24h, 48h and after
96h). After the last recording the mice were sacrificed and the following organs are
removed and prepared: Skin, blood, spleen, liver and lungs.
BFI was performed on a In Vivo Imaging System and analyzed on a computer
equipped with Living Image Software. After anesthetizing the mice with 5%
Isoflurane ((RS)-Difluormethoxy-1-chlor- 2,2,2-trifluorethan) the mice were placedin the recording chamber. The excitation and emission maxima of the IR-dye were
determined in previous experiment as 745nm and 820nm. Various exposure time
spans were applied starting with one second. After the first recording the remaining
NP from the injection in the tail was covered and further pictures were taken.
Usually an excitation time of five seconds was applied but other values are used
as well which is further indicated in the text. For all measurements Binning (CCD
resolution) was set to 8 and F/Stop (Aperture) to 1, the subject height was set to
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1.50 cm. The imaging mode used was fluorescent / photograph.
For statistical analysis the radiant efficiency of the regions of interest (ROI) was
determined :
The Radiant efficiency is a the ratio between the power emitted by a source of
radiation to the power consumed by it For each animal the liver region, the lung
regions and the region of the snout were measured
3. Nomenclature, Abbreviations and Units
Emission light ( photons/sec/cm2/str)
Radiant Efficiency n = ( )
Excitation light ( W/cm2 )
4. Results
4.1 In VitroExamination of DCs loaded with NP
For the in vitro studies DCs were generated, then supplemented and incubated
with DCs as described in the methods section. The cells were examined via FACS
analysis followed up by a statistical evaluation of the obtained results. As an
image-guided approach an additional cLSM analysis was performed. Each NP was
tested in four different concentrations (25, 75, 150, 300 g/ml) with an additional
negative control. The viability of the cells and their NP uptake were estimated by
FACS analysis. The cLSM measurements supplied additional information with
regard to localization and behavior of the NP and of the DCs
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4.1.1 Viability determination of DCs
For the determination of the DC viability the DCs were stained with APC
conjugated monoclonal anti-CD11c and 7AAD. The obtained FACS data was gated
for CD11c positive and 7AAD negative populations (CD11cpos/7AADneg) as
exemplified in Fig.4.1.1. The figure shows a representative measurement of NP
GB-PS-62 [75 g/ml] loaded DCs
For each gate it is possible to quantify the contained events. For this purpose the
a. b.
c. d.
Fig.4.1.1 : Evaluation of the obtained FACS data. The blots show theexemplified results of the APC and 7AAD measurement comparing a negativecontrol sample (a. and b.) to an APC/7AAD stained sample (c.) and (d.). On the lefthand side APC intensity on the y-axis is plotted vs. SSC intensity on the x-axis.Each dot represents a recorded event. On the right hand side the x-axis representsthe intensity of the 7AAD signal while the y-axis represents the according amountof measured events. (a.) and (b.) show the results of a negative control sample.The cells were stained only with APC-conjugated IgG (isotype control). The mainpopulations were gated s negative while events outside these gates were regardedas positive events for the according parameter. (c.) and (d.) show the results of aDC sample stained with CD11c and 7AAD. In (c.) the main population is located inquadrant A1 / A2 and hence CD11cpos. These cells were gated as DCs. (d.) Thehistogram only views the events of the gate DCs. The first peak represents7AADneg cells (as determined in (b.)) gated as viable. The remaining events are7AAD positive. The second peak is gated as Apoptotic and the third peak is gated
as Dead
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number of events within a gate is determined. On the one hand the result can be
estimated as the absolute number of events. On the other hand, it is possible to
express the result either as the percentage of the total number of events or as the
percentage of the number of gated events displayed in the plot. For the FACS
measurements displayed in Fig.4.1.1 the obtained data is presented in in Tab 4.1.1.
Gate Number of events % Total % Gated
DCs 8036 80.36 80.36
Viable 7392 73.92 91.99
Apoptotic 246 2.46 3.06
Dead 398 3.98 4.95
Total events 10000 100.00 100.00
In our FACS measurements for each sample tube a total number of 104 events
was recorded analyzed. Populations that were gated during the analysis can be
directly compared to this number (% Total). The resulting figures are absolute and
not altered by any gate of higher order. In contrast to this the number of events can
be compared to the gate of the next higher order (% Gated). For example the gate
DCs was created in a plot displaying all events. In this case the gate of next
higher order is the total number of events resulting in the same values for % Total
and % Gated.
% Total (DCs) = Number of events (DCs) / Total events = % Gated (DCs)
= 8036 events / 10000 events = 0.8063
= 80.36 %
In fact 80.36 % of the total events were regarded as CD11c positive and gates as
DCs.
Tab 4.1.1 : Quantification of the events gated in Fig.4.1.1. An overview of thenumber of events contained by gates presented in Fig.4.1.1 (c.) and (d.) Theamount of events can be displayed as a total number of events as well as afraction of either the total number of events measured (% Total) or as a fraction ofthe gate that is displayed in a plot (% Gated)
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The gates Viable, Apoptotic and Dead were created in a plot that only displays
the gate DCs assigning it as the gate of higher order. In this case % Total and %
Gated deliver different results.
% Total (Viable) = Number of events (Viable) / Total events
= 7392 events / 10000 events = 0.7392
= 73.92 %
% Gated (Viable) = Number of events (Viable) / Number of events (DCs)
= 7392 events / 8036 events = .9199
= 91.99 %
While 73.92 % of to the total number of events were 7AADneg and gated as viable,
91.99 % of the cells gated as DCs were contained in this gate.
To determine the relative DC viability the FACS data was gated as described
above as viable, apoptotic and dead. The results were determined as % Gated for
each population. The influence of NP on the viability was estimated by comparing
the results of samples incubated with increasing concentrations of NP as
described above to a control sample without NP that was stained with anti-CD11c
and 7AAD. The maintained data was analyzed and displayed with the help of
Microsoft Excel.
The complete set of data concerning the DC viability from this experiment is
presented in Tab.4.1.2 and is visualized in Fig.4.1.2
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Fig.4.1.2 : Vitality of DCs in dependence on GB-PS62 concentration. This figure visualizes the data ofTab.4.1.2 and shows vital, apoptotic and dead cellpercentage in relation to the NP concentration.
Probes viable apoptotic dead
NC (negative control) 92.22 3.81 3.98
DC+GB-PS 62 [25 g/ml] 91.89 2.39 5.74
DC+GB-PS 62 [75 g/ml] 92.32 2.71 4.95
DC+GB-PS 62 [150 g/ml] 90.58 2.32 7.09
DC+GB-PS 62 [300 g/ml] 87.45 4.34 8.20
The viability remains constant at low concentrations of GB-PS 62 at about 92 %. At
concentrations above
75 g/ml the viability
starts to drop and the
amount of dead cells
begins to rise. At the
highest concentrationthe viability rates has
lost roughly 5 % and
the percentage of
dead cells has
doubled from ~4 % to
~8 %. Also the level of
apoptotic cells at 300 g/ml is above the levels of the other concentrations but still
at the same level as the NC.
A slight decrease in viability was estimated for the highest concentrations of GB-
PS 62 (150 and 300 g/ml), while the percentage of dead cells increased at these
concentrations. At lower dosages of NP 62 the viability exhibits only minor
variations while the level of apoptotic cells does not surpass that of the negative
control (NC) at any concentration.
Tab 4.1.2: Vitality of DCs incubated with NP GB-PS 62. Viable, apoptotic andDead cells as % Gated of the CD11cpos population. The negative control sample(NC) shows the vitality of DCs without the addition of NP whereas the othersamples represent the vitality at increasing levels of NP (25, 75, 150 and 300g/ml).
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4.1.2 Determination of NP uptake into DCs
The second aspect to be analyzed in ourin vitro studies was the uptake of NP into
DCs. To determine the presence of NP in DCs during FACS measurements we
stimulated the fluorescence of BODIPY contained in the NP. The corresponding
signal was detected in the fluorescence-1 channel (FL1 or FITC channel, filter
530/30 nm) of the cytometer. The NP uptake was estimated as % Gated of the
population of viable DCs that was determined in 4.1.1. Cells showing an increased
signal compared to a negative sample were regarded as NPpos as exemplified in
Fig.4.1.3
.
While the negative control only shows a very weak fluorescent signal a strong
increase of the fluorescence intensity can be seen in the GB-PS 62 containing
sample. For the statistical determination of particle uptake the median
fluorescence intensity (MFI) of the NP positive population was estimated for each
sample and the results of the different concentrations were compared to each
other.
a. b.
Fig.4.1.3 : FL1 signals of loaded and unloaded DCs. (a.) shows the FL1 signalof a negative control sample. Events below the intensity peak were gated as NPnegative; all events above were gated as NP positive. (b.) shows the signal ofDCs that were incubated with 300 g/ml GB-PS 62. 93 % of the events wereestimated as NP ositive.
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Fig.4.1.4 : MFI of cells gated as NPpos. The
signal intensity correlates with the particleconcentration
Probes MFI % Gated
NC 0.7 0.27
DC+GB-PS 62 [25 g/ml] 5.1 88.03
DC+GB-PS 62 [75 g/ml] 4.9 87.63
DC+GB-PS 62 [150 g/ml] 6.2 88.96
DC+GB-PS 62 [300 g/ml] 6.8 92.41
In Tab.4.1.3 the MFI at an increasing dosage of NP 62 is presented. The MFI
jumps up with the addition of NP 62 compared to the NC sample. With increasing
particle concentration the MFI shows a trend of increasing proportionally, easier to
be spotted in Fig.4.1.4 which interprets the MFI data graphically. Tab.4.1.3 also
shows that for all particle containing samples, roughly 90% of the cells have taken
up NP. This rate is very similar at all concentration although there is a slight
increase at the highest concentration.
The gating strategy reviewed in this chapter and in chapter 4.1.1 was applied in
the following experiments to quantify DC viability and particle uptake. Although the
results shown represent onlya single approach, the
following experiments were
conducted in a duplicate
approach under identical
conditions. For these
experiments the statistical
mean and the standard
deviation of the according
results were determined and
used for further evaluation.
4.1.3 cLSM results for NP GB-PS 62
Additionally to the FACS based approach we monitored the NP behavior on a
cellular level by the means of cLSM. This part of the experiment is intended to be a
Tab 4.1.3 : GB-PS 62 MFI and %Gated of the NP positive fraction. Thesamples contain an increasing amount of NP 62 in addition to a negative controlsample (NC).
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Fig.4.1.5 : cLSM image of mDCs with incorporated GB-PS 62 [25 g/ml]. Theparticles contain BODIPY fluorescent dye (green). Cell membrane stain (red) wasperformed with CellMask Orange. Hoechst 33342 (blue) was used for nuclearstaining. The different channels recorded are shown separately in pictures (A) to(D). An overlay of the fluorescence channels is given in (E).
graphical interface for optical analysis of the NP containing cells. Even though
there is no quantitative breakdown, the obtained recordings can deliver
interpretable and very illuminating results. As for the FACS analysis the NP was
tested on DCs in four different concentrations, following the same protocol for the
NP incorporation.
The analysis via LSM can reveal certain aspects that are not covered by the by the
means of flow cytometry. Hence it is possible to determine particle localizattion
and the actual incorporation into the cells. As shown in Fig.4.1.5 the green
fluorecent signals indicate large quantities of NP loaded with BODIPY. The staining
of the plasma membrane of the DCs with CellMask Orange (red) and the
nuclear staining with Hoechst 33342 (blue) have worked well and are clearly
visible. The NP is mainly present in clusters located and also some smaller
aggregates within the cells, extracellular particle is not visible. A smaller portion of
A B C
D E
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NP returns a yellow signal in the overlay views (E) top left corner, resulting from
the overlap of the green fluorescent signal of the particle and the red stained cell
membrane. This effect can be seen if particle is attatched to the plasma membrane
of a cell.
Results from the application of different particle conentrations on DCs is diplayed
in Fig.4.1.6. The negative control sample (A) shows the typical morphology of
mature DCs.
Notice the dendrites deriving from the plasma membrane. At the lowest
concentration (B) tested the green fluorescent signal of the particle is clearly
visible. At 75 g/ml (C) the increased amount of NP is reflected in the higher
amounts of NP present in the cells. The particle also shows an increased tendency
towards aggregate formation. Also there is a slight color shift detectable at the
particle cluster. For the highest concentrations (D,E) this is especially visible.
Although the fluorescent signal is still evident in the green channel (not shown),
the signal from the internalized NP appears as light blue in the overlay view.
A B C
D E
Fig.4.1.6 : cLSM image series of mature DCs with increasingconcentration of incorporated GB-PS 62. (A) negative control; (B) 25 g/ml;(C) 75 g/ml (D) 150 g/ml (E) 300 g/ml.
B
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4.1.4 Results NP 61
As described above for NP 62 the particle GB-PS 61 was tested at 25. 75, 150 and
300 g/ml. The FACS data was analyzed for vitality and particle uptake with the
following results.
Sample viable apoptotic dead
NC 90.06 6.58 3.33
DC+GB-PS 61 [25 g/ml] 87.20 7.27 5.54
DC+GB-PS 61 [75 g/ml] 76.30 14.07 9.69
DC+GB-PS 61 [150 g/ml] 65.43 18.87 15.74
DC+GB-PS 61 [300 g/ml] 66.91 23.29 9.66
Sample STDN (%) viable apop. dead
NC 1.49 7.07 27.63
DC+GB-PS 61 [25 g/ml] 3.10 32.46 6.32
DC+GB-PS 61 [75 g/ml] 3.39 3.41 21.78
DC+GB-PS 61 [150 g/ml] 3.84 0.45 15.60
DC+GB-PS 61 [300 g/ml] 8.27 12.84 28.53
Sample MFI +/- STDN % Gated NPpos +/- STDN
NC 1.7 0.1 1.86 0.93
DC+GB-PS 61 [25 g/ml] 2.1 0.1 16.63 3.17
DC+GB-PS 61 [75 g/ml] 2.9 0.0 43.87 0.00
DC+GB-PS 61 [150 g/ml] 2.5 0.1 62.38 1.53
DC+GB-PS 61 [300 g/ml] 2.6 0.1 76.30 0.92
Tab 4.1.4 : Vitality of mature DCs after incubation with GB-PS 61. Viability,apoptotic and dead cells as % Gated of CD11cpos cells for increasingconcentrations of NP61. (Average from duplicated approach, STDN given inTab.4.1.5
Tab 4.1.5 : Standard deviations (%) for the results presented in Tab.4.1.4.
Tab 4.1.6 : GB-PS 61 uptake.Amount of cells gated as GB-PS 61 positive andMFI with the according standard deviations (STDN) for all concentrations.
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The obtained data was blotted in Fig.4.1.7
Regarding Fig.4.1.7 (A) it becomes evident that the cell vitality is altered with
increasing concentration of NP 61. The viability is at about 90 % for the untreated
DCs. While remaining more or less constant at 25 g/ml GB-PS 61, a break-in can
be observed at 75 g/ml. The viability falls below 80% and drops roughly the by
the same amount at 150 g/ml. At the maximum concentration the viability remains
at this level. The amount of apoptotic cells is slightly higher than the amount ofdead cells at all concentrations.
For the particle uptake Fig.4.1.7 (B) an increased rate was determined as the NP
was introduced at the lowest concentration. A maximum value is reached at 75
g/ml. At higher concentrations the uptake rate stagnates and even shows a slight
loss.
Fig.4.1.7 : DC Vitality and NP Uptake in dependence on particleconcentration. The Figure shows the amount of viable, apoptotic and dead cells(A) and the estimated rate of uptake (B) at increasing rates of NP concentrationcompared to a negative control sample (NC).
A B
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The particle GB-PS 61 was our first object we investigated via LSM analysis.
Surprisingly the findings have shown some abnormalities concerning the
fluorescence color spectrum, which can be seen in Fig.4.1.8. While the staining did
work out well for the negative control, all images containing NP view various colors,
untypical for the particle
and the applied staining.
Besides the altered color
(white / light blue / pink) of
the NP derived
fluorescence signal, the
increase of particle
concentration is clearly
evident the image series.
The NP gives a strong
signal and tends to form
A B C
D E
Fig.4.1.8 : cLSM image series of mature DCs with increasing concentrationof incorporated GB-PS 61. (A) negative control; (B) 25 g/ml; (C) 75 g/ml (D)150 g/ml (E) 300 g/ml.
Fig.4.1.9 : Split channel view of DCs containing GB-
PS 61 [300 g/ml]
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large aggregates. The split channels view of image (E) is displayed in Fig.4.1.9.
Although it becomes clear that the color shift is due to an overlap of the green
BODIPY signal with the red and blue signal from membrane and core staining, this
phenomenon requires further dispute that will be attended during the discussion.
4.1.5 Results NP 63
For NP 63 only the uptake is presented as the 7AAD measurement was showing
distortions that could not be compensated for. As shown in Fig.4.1.10 the particle
uptake increased until a concentration of 150 g/ml was reached, then it stabilized.
The obtained data for the uptake is also displayed in Tab.4.1.7.
Probes MFI +/- STD % Gated NPpos +/- STD
NC 1.7 0.2 1.31 1.31
DC+GB-PS 63 [25 g/ml] 5.5 2.1 67.91 67.91
DC+GB-PS 63 [75 g/ml] 15.8 1.1 90.07 90.07
DC+GB-PS 63 [150 g/ml] 18.9 1.3 94.70 94.70
DC+GB-PS 63 [300 g/ml] 19.2 0.2 99.32 99.32
Fig.4.1.10 : Uptake of GB-PS 63 into DCs at incresing levels of particle
concentration. The MFI determined for NP 63 at all tested concentrations
Tab.4.1.7 : GB-PS 63 uptake.Amount of cells gated as GB-PS 63 positive andMFI with the according standard deviations (STDN) for all concentrations.
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The LSM analysis of particle GB-PS 63 shows alteration in the color spectrum
similar to that observed for NP 61 despite at the higher concentrations pink is the
predominant color for NP 63. Again the color shift only occurs at higher
concentrations of NP. Anyways, the increase in particle concentration is visible
comparing the images and also the staining worked well. This particle as well
shows the trend of forming agglomerations. There is much particle, especially
larger aggregates, located outside of the cells.
4.1.6 Results NP 59
The particle BR 59 was tested in the concentrations 25, 75, 150 and 300 g/ml via
FACS measurement and cLSM imaging. The vitality and the uptake of NP were
determined for the DCs. The LSM images were qualitatively analyzed.
A B C
D E
Fig.4.1.11 : cLSM image series of mature DCs with increasingconcentration of incorporated GB-PS 61. (A) negative control; (B) 25 g/ml; (C)75 g/ml (D) 150 g/ml (E) 300 g/ml.
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Probes viable apoptotic dead
NC 80.47 14.40 5.06
DC+ BR 59 [25 g/ml] 77.78 16.32 5.83
DC+ BR 59 [75 g/ml] 81.40 13.04 5.49
DC+ BR 59 [150 g/ml] 80.20 15.19 4.43
DC+ BR 59 [300 g/ml] 70.77 24.59 3.90
Probes STDN (%) viable apoptotic dead
NC 0.00 0.00 0.00
DC+ BR 59 [25 g/ml] 2.70 21.79 24.70
DC+ BR 59 [75 g/ml] 2.13 13.96 0.91
DC+ BR 59 [150 g/ml] 1.20 0.49 21.90
DC+ BR 59 [300 g/ml] 20.72 51.48 33.50
Probes MFI STDN% GatedNPpos STDN
NC 2.30 0.20 0.54 0.08
DC+ BR 59 [25 g/ml] 5.10 0.50 49.75 5.08
DC+ BR 59 [75 g/ml] 21.85 6.75 73.95 5.21
DC+ BR 59 [150 g/ml] 31.25 0.85 86.08 0.83
DC+ BR 59 [300 g/ml] 130.60 21.50 89.69 1.36
The obtained data is presented as a blot in Fig.4.1.12. (A) The figure shows that
the particle BR 59 has no toxic effect on the tested DCs, only a slight decrease at
the highest concentration is visible but it has to be considered that there was a
high discrepancy between the obtained results at this concentration (see
Tab.4.1.11.). (B) The uptake rate increases considerably at all concentrations but a
Tab.4.1.10 : Vitality of mature DCs after incubation with GB-PS 61. Viability,apoptotic and dead cells as % Gated of CD11cpos cells at increasingconcentrations of NP BR 59. (Average from duplicated approach, STDN given inTab.4.1.11)
Tab.4.1.12 : BR 59 uptake into DCs.Amount of cells gated as BR 59 positiveand MFI with the according standard deviations (STDN) for all concentrations
Tab.4.1.11 : Standard deviation for the results presented in Tab.4.1.10
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relatively big jump can be observed at the highest concentration.
In contrast to the other LSM measurements BR 59 was tested using DRAQ5
(Biostatus) was used as a nuclear stain. Fig.4.1.13 shows that the particle showed
Fig.4.1.12 : DC vitality and uptake of BR 59 in dependence on NPconcentration. The Figure shows the amount of viable, apoptotic and deadcells (A) and the estimated rate of particle uptake (B) at increasing rates of NPconcentration compared to a negative control sample (NC).
A B
Fig.4.1.14 : cLSM Z-Stack analysis of DCs with BR 59. The image seriesshows the same location with increasing depths.
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less aggregation compared to the other particles and that there was no color
distortion. At the low concentrations shown in (B) and (C) only few cells prove to
have incorporated particle. At 150 g/ml (D) most cells have taken up NP in small
amounts. Like in Fig.4.1.12 (B) the LSM images shows a huge increase in uptake
at the highest concentration (E).
BR 59 revealed a tendency to locate near the nucleus in this experiment. In some
cases the signal was partially obliterated by the nuclear staining which became
evident in the split channel view (not shown here). Fig.4.1.14 shows a series of
images of two cells with incorporated NP with an increasing depth. In both cells the
particle is located around the nuclear region.
A B C
D E
Fig.4.1.15 : cLSM image series of mature DCs with increasingconcentration of incorporated BR 59.A) negative control; (B) 25 g/ml; (C) 75g/ml (D) 150 g/ml (E) 300 g/ml. The nuclear staining was performed usingDRAQ5
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4.2 Biodistr ib ut ion of NP in a Mouse model
Beneath the in vitro evaluation of the Nanoparticles we were interested in the bio
distribution of the particles in a model organism. To study the in vivo behavior of
the different NPs we intravenously injected predefined particle dosages and
monitored the particle distribution at different time points after the injection by the
means of BFI. Besides optical analysis of the obtained images, BFI allows to
quantify the measured emission at user defined regions of interest (ROIs).
4.2.1 Mouse kinetics
During this experiment we analyzed the fluorescence intensity at different locations
where we expected to find an increased signal caused by the presence of IR-dye
780 which is contained in the NPs. Examinations of the test animals were
performed at 0h, 4h, 24h, 48h and 96h after particle injection. The emission at the
liver and lung regions as well as the emission at region of the snout was evaluated.
During the measurements the site of infection was covered to avoid the presence
of unspecific signal caused by retained NP. All particles were administered at a
concentration of 3.7 % (v/v)
A B C
ED
Fig.4.2.1 : Bio distribution of BR 59 at different time points after theinjection. The images show the radiant efficiency measured at 0h (A), 4h (B), 24h(C), 48h (D) and 96h (E) after administration of NP 59 (3,7%). While mouse (1),(2) and (3) were treated with NP, mouse (4) did not receive a particle injection andwas used as reference.
1 2 3 4
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As shown in in Fig.4.2.1 for the mice treated with BR 59 a signal (colored spots) is
returned indicating the presence of NP while no signal was detected for the
untreated reference subject. The strength of the emitted radiance can be deduced
from the color scale on the right hand side of the Figure. The main source of
emission is located around the liver region and at the snout of the subjects. Amaximum of the signal intensity is evident immediately after NP application. Four
hours after the injection the emission from the particle started to cease. Although
mouse (1) and (3) still bore a strong signal at liver region, the area of emission
decreased. The measured radiance further decreased during the following
measurements at 24h, 48h and 96h. Anyway, the emittance is still detectable after
96h for all of the NP treated mice. The strongest signal was returned from mouse
(1) in all measurements. The lower signal in the other mice (2 and 3) is due to aless successful injection of the particle (see Tab.4.2.5) as it is not always possible
to hit the vein with the complete NP dosage.
The measured radiant efficiency for the specified ROIs is presented in Tab.4.2.1.
An illustration of the obtained data is given at the end of this chapter in comparison
to the results collected from approaches with the remaining NPs.
The results from the application of GB-PS 61 can be seen in Fig.4.2.2 and
Tab.4.2.2. The particle showed a very strong signal while the control mouse did not
show any signal. The signal is mainly located at the liver, lung and snout regions
NP 59 time (h) 0 4 24 48 96
Mouse 1 Snout 8.310E+06 8.490E+06 1.075E+07 9.429E+06 9.290E+06
Lung L 1.042E+07 9.227E+06 1.036E+07 1.033E+07 8.581E+06
Lung R 8.491E+06 9.573E+06 9.736E+06 8.685E+06 8.462E+06
Liver 1.784E+07 1.489E+07 1.244E+07 1.228E+07 1.069E+07
Mouse 2 Snout 7.016E+06 7.348E+06 8.546E+06 8.319E+06 8.472E+06
Lung L 7.387E+06 7.452E+06 8.131E+06 7.949E+06 7.619E+06
Lung R 7.610E+06 7.451E+06 7.583E+06 7.864E+06 7.498E+06Liver 1.454E+07 1.098E+07 1.087E+07 1.098E+07 1.061E+07
Mouse 3 Snout 6.819E+06 8.498E+06 8.879E+06 8.973E+06 8.570E+06
Lung L 7.400E+06 7.381E+06 8.455E+06 8.205E+06 7.607E+06
Lung R 7.223E+06 7.474E+06 7.460E+06 7.307E+06 7.346E+06
Liver 1.383E+07 1.249E+07 1.059E+07 1.034E+07 1.082E+07
Control Snout 5.917E+06 6.012E+06 6.687E+06 6.389E+06 6.168E+06
Lung L 5.602E+06 6.281E+06 6.075E+06 5.949E+06 6.217E+06
Lung R 5.407E+06 6.180E+06 6.102E+06 6.027E+06 6.257E+06
Liver 5.795E+06 6.642E+06 6.742E+06 6.563E+06 6.860E+06
Tab.4.2.1 : Radiant Efficiency after the intravenous injection of BR 59 (3,7%).The radiant efficiency was determined after 0h, 4h, 24h, 48h and 96h for the ROIs
defined for Snout, Liver as well as for left and right lung
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but also around the fore- and hind legs and in some cases almost over the entire
body ((B) and (C) mouse 1). The source of the highest radiant efficiency is the liver
region. The signal reached its maximum intensity after four hours after the injection
with almost no decrease even after 96h. The injection worked very well for this
particle, the whole dosage could be applied for all mice (Tab.4.2.5).
A B C
ED
1 2 3 4
Fig.4.2.2 : Bio distribution of GB-PS 61 at different time points after theinjection. The images show the radiant efficiency measured at 0h (A), 4h (B), 24h(C), 48h (D) and 96h (E) after administration of NP 61 (3,7%). While mouse (1),(2) and (3) were treated with NP, mouse (4) did not receive a particle injection andwas used as reference.
Tab.4.2.2 : Radiant Efficiency after the intravenous injection of GB-PS 61(3,7%). The radiant efficiency was determined after 0h, 4h, 24h, 48h and 96h forthe ROIs defined for Snout, Liver as well as for left and right lung
NP 61 time (h) 0 4 24 48 96
Mouse 1 Snout 1.539E+07 2.681E+07 2.302E+07 2.502E+07 2.078E+07
Lung L 2.752E+07 2.364E+07 1.885E+07 2.655E+07 1.930E+07
Lung R 1.205E+07 2.011E+07 2.097E+07 1.548E+07 1.545E+07
Liver 1.540E+07 3.122E+07 2.570E+07 2.610E+07 2.563E+07
Mouse 2 Snout 1.721E+07 1.488E+07 1.448E+07 1.325E+07 1.246E+07
Lung L 1.904E+07 1.836E+07 1.179E+07 1.549E+07 1.527E+07
Lung R 1.349E+07 1.170E+07 1.548E+07 1.227E+07 1.129E+07
Liver 1.244E+07 1.625E+07 1.630E+07 1.693E+07 1.712E+07
Mouse 3 Snout 1.419E+07 1.559E+07 1.306E+07 1.294E+07 1.257E+07
Lung L 1.712E+07 1.380E+07 1.361E+07 1.724E+07 1.380E+07
Lung R 9.801E+06 1.232E+07 1.335E+07 1.102E+07 1.054E+07
Liver 1.058E+07 1.468E+07 1.517E+07 1.544E+07 1.530E+07
Control Snout 6.068E+06 6.073E+06 6.837E+06 6.108E+06 6.246E+06
Lung L 5.565E+06 6.115E+06 6.677E+06 6.022E+06 6.289E+06
Lung R 5.467E+06 5.989E+06 6.349E+06 5.489E+06 6.033E+06
Liver 5.610E+06 6.196E+06 6.667E+06 6.094E+06 6.339E+06
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The bio distribution study of GB-PS 62 shown in Fig.4.2.3 only revealed a signal
for one of the mice (2) due to low a injection efficiency regarding mouse (1) and
mouse (3) (see Tab.4.2.5). The signal is visible exclusively at the liver region. The
signal strength is maintained at a constant level over the whole period of 96h. In
Fig.4.2.3 : Bio distribution of GB-PS 62 at different time points after theinjection. The images show the radiant efficiency measured at 0h (A), 4h (B), 24h(C), 48h (D) and 96h (E) after administration of NP 62 (3,7%). While mouse (1),(2) and (3) were treated with NP, mouse (4) did not receive a particle injection andwas used as reference.
Tab.4.2.3 : Radiant Efficiency after the intravenous injection of GB-PS 62(3,7%). The radiant efficiency was determined after 0h, 4h, 24h, 48h and 96h forthe ROIs defined for Snout, Liver as well as for left and right lung
NP 62 time (h) 0 4 24 48 96
Mouse 1 Snout 6.864E+06 1.014E+07 1.658E+07 1.353E+07 1.089E+07
Lung L 7.430E+06 7.486E+06 9.273E+06 9.378E+06 8.056E+06
Lung R 6.563E+06 8.326E+06 9.536E+06 9.509E+06 8.326E+06
Liver 8.482E+06 8.787E+06 9.733E+06 9.237E+06 8.677E+06
Mouse 2 Snout 1.091E+07 1.657E+07 1.763E+07 1.698E+07 1.534E+07Lung L 1.304E+07 1.410E+07 1.415E+07 1.434E+07 1.350E+07
Lung R 1.179E+07 1.137E+07 1.264E+07 1.289E+07 1.297E+07
Liver 5.117E+07 3.820E+07 3.558E+07 3.837E+07 4.888E+07
Mouse 3 Snout 6.081E+06 9.152E+06 1.634E+07 9.548E+06 1.254E+07
Lung L 6.259E+06 6.753E+06 1.063E+07 1.016E+07 9.755E+06
Lung R 5.723E+06 6.573E+06 9.079E+06 1.124E+07 9.049E+06
Liver 6.558E+06 7.430E+06 9.734E+06 1.062E+07 1.025E+07
Control Snout 6.051E+06 6.598E+06 7.102E+06 6.732E+06 7.121E+06
Lung L 6.225E+06 6.215E+06 6.229E+06 6.222E+06 6.467E+06
Lung R 5.825E+06 6.235E+06 6.197E+06 6.046E+06 6.538E+06
Liver 6.127E+06 6.526E+06 6.916E+06 6.300E+06 6.769E+06
A B C
ED
1 2 3 4
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Tab.4.2.3 the radiant efficiency for the measured ROIs is listed.
The last particle to be tested is GB-PS 63. As shown in Fig.4.2.4. NP 63 showed
the strongest signal among the tested particles even though the injection efficiency
for all mice was only about 50 % (see Tab.4.2.5). Beneath covering most parts of
the body the area with the highest signal strength is located in the liver and lung
regions while the control mouse did not show any visible signal.
A B C
ED
1 2 3 4
Fig.4.2.4 : Bio distribution of GB-PS 63 at different time points after theinjection. The images show the radiant efficiency measured at 0h (A), 4h (B), 24h(C), 48h (D) and 96h (E) after administration of NP 63 (3,7%). While mouse (1), (2)
and (3) were treated with NP, mouse (4) did not receive a particle injection andwas used as reference.
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During the whole series the measured radiant efficiency only viewed minor
variations and after 96h particle was still present. The results for all ROIs are listed
in Tab.4.2.4.
For the further evaluation of the collected data the results were summarized as
NP 63 time (h) 0 4 24 48 96
Mouse 1 Snout 2.458E+07 2.303E+07 3.136E+07 2.770E+07 2.214E+07
Lung L 4.044E+07 3.849E+07 4.128E+07 2.480E+07 2.889E+07
Lung R 2.202E+07 2.358E+07 2.653E+07 2.825E+07 2.417E+07
Liver 2.385E+07 2.484E+07 2.698E+07 2.709E+07 2.583E+07
Mouse 2 Snout 1.454E+07 1.119E+07 9.287E+06 1.168E+07 1.077E+07
Lung L 2.483E+07 1.979E+07 1.950E+07 1.786E+07 1.807E+07
Lung R 1.260E+07 1.409E+07 1.336E+07 1.183E+07 1.232E+07
Liver 1.652E+07 1.803E+07 1.849E+07 1.904E+07 1.768E+07
Mouse 3 Snout 2.095E+07 2.005E+07 2.386E+07 2.123E+07 1.986E+07
Lung L 1.768E+07 2.475E+07 2.457E+07 2.203E+07 2.081E+07
Lung R 1.859E+07 1.915E+07 2.220E+07 2.307E+07 1.837E+07
Liver 1.616E+07 2.303E+07 2.215E+07 2.304E+07 2.291E+07
Control Snout 5.791E+06 6.451E+06 6.984E+06 6.394E+06 7.092E+06
Lung L 5.474E+06 6.571E+06 6.042E+06 5.668E+06 6.689E+06
Lung R 5.365E+06 5.605E+06 5.933E+06 5.744E+06 6.540E+06
Liver 5.800E+06 6.408E+06 6.483E+06 6.243E+06 6.812E+06
Tab.4.2.4 : Radiant Efficiency after the intravenous injection of GB-PS 63(3,7%). The radiant efficiency was determined after 0h, 4h, 24h, 48h and 96h forthe ROIs defined for Snout, Liver as well as for left and right lung
Fig.4.2.5 : Particle distribution analysis, overview of all measurements. Eachblot displays the radiant efficiency for one ROI (averaged results) at 0h, 4h, 24h,48h and 96h after NP application. The colors indicate the NP (red = control,purple = NP 59, dark blue = NP 61, light blue = NP 62, green = NP 63)
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shown in Fig.4.2.5. The blots view the average radiant efficiency development of
the ROIs Liver, Snout and Lung, comparing the tested NP. All particles showed
increased values compared to the negative control. The strongest signal was
found for NP 63 in 80 % of all ROIs and time points. The negative control showed
constant radiant efficiency values slightly above 0.500E7
for all ROIs. At the liverregion all NPs showed elevated radiant efficiency levels with at least 2.5 times the
intensity of the negative control signal. For the GB-PS particles the radiant
efficiency was even higher, all three particles showed signals with about four times
the intensity of the negative control over the whole time span. Only the GB-PS 61
signal was lower at 0h, but had reached a similar strength to NP 62 and NP 63
after 4h. The emittance of BR 59 was weaker than the emittance measured for the
GB-PS particles and the signal strength decreased constantly. At the end theradiant efficiency was about twice as high as for the reference.
Regarding the snout region the particles GB-PS 61 and GB-PS 63 showed a
constant signal with similar strength compared to the liver region. NP 62 showed a
slightly increased emittance at 0h compared to the negative control. The signal
ascended after 4h reaching a maximum at 24h, close to the signal strength of the
other GB-PS particles, and then evens out. For BR 59 the values determined for
the snout region were all close to the negative control.
In the lung region two of the particles revealed a strong signal that remained
constant over the whole 96 hours: GB-PS 63 had the highest radiant efficiency
between four and five times as high as the negative control. The second particle
was GB-PS 61, although the emission was not as strong as for NP 63, it was still
three times higher than the negative control. Little to no signal was returned from
the other particles, only NP 62 reached a threshold doubling the negative control
after 24h.
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NPInjection Efficiency
(%)Injection se-
quence Date of birth
61 100 1 05/01/2011
61 100 2 29/12/2010
61 100 3 29/12/2010
62 50 1 05/01/2011
62 50 2 29/12/2010
62 20 3 29/12/2010
63 50 1 29/12/2010
63 50 2 03/11/2010
63 50 3 29/12/2010
59 100 1 29/12/2010
59 50 2 03/11/2010
59 50 3 29/12/2010
Control - 1 29/12/2010
4.2.2 Organ distribution
After 96h the mice were sacrificed, liver, lungs, spleen and a skin sample were
removed. Also a blood probe was taken. The results of the BFI analysis are
presented in Fig.4.2.6 and Fig.4.2.7. The original images and the raw data are
shown in Fig.4.2.8 and Tab.4.2.6.
The findings displayed in Fig.4.2.6 show the particle distribution for the examinedorgans indicated by the increase of radiant efficiency. The particle presence
detected in the skin is displayed in Fig.4.2.7. The radiant efficiency measured for
the blood samples was subtracted from the values obtained for the skin, in order to
compensate for the emission from blood present in the skin capillary vessels.
While all particles were detectable in the liver, the distribution to the other organs
varied from particle to particle. The NP GP-PS 61 showed an intense presence in
lung (strongest signal) and liver. A particle contamination was also detected in the
spleen and NP traces were found in the skin as well, especially for one animal.
There was no indication of particle remains in the blood. For the particle GB-PS 62
only one of the mice showed a main increase in radiant efficiency. The signal
indicated particle presence mainly in the liver but also in the lung. An increased
emittance was also detected in the spleen for this animal, while there was no
increased intensity visible in the blood. Anyway, all subjects showed an increased
emission in the skin. The distribution analysis of GB-PS 63 revealed that this
Tab.4.2.5 : Injection efficiency and order.
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particle was mainly located in the lung after 96h. Also the liver and spleen showed
strong signs of contamination and an emission peak was detected in the skin for
mouse 1 and mouse 3. Although barely visible in the blots due to its low intensity,
NP 63 was the only particle that returned a signal in the BFI investigation of the
blood samples (see Fig.4.2.8 E). The analysis of the data obtained for BR 59demonstrated that this particle is
mainly deposited in the liver. A contamination of lung, spleen and skin is detectable
as well but measured emittance was very low compared to the liver signal. A
particle presence in the blood samples could not be confirmed.
Fig.4.2.6 : Particle distribution to the examined organs after 96h. Each blotshows the results obtained for one particle. All tested mice are displayedcompared to a NP negative control (purple).
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0,000E+00
0,200E+04
0,400E+04
0,600E+04
0,800E+04
1,000E+04
1,200E+04
1,400E+04
1,600E+04
NP61 NP62 NP63 NP59
Emission
Nanoparticle
Skin (Blood substracted) after 96h
Mouse 1
Mouse 2
Mouse 3
Control
Subject Blood Liver Spleen Skin Lung NP
Mouse 1 4.607E+06 8.709E+07 4.227E+07 1.836E+07 9.152E+07 NP61
Mouse 2 4.047E+06 6.176E+07 3.608E+07 8.232E+06 8.430E+07 NP61
Mouse 3 3.838E+06 6.324E+07 2.185E+07 7.028E+06 1.547E+08 NP61
Mouse 4 3.668E+06 1.213E+07 5.632E+06 8.339E+06 7.329E+06 NP62
Mouse 5 3.785E+06 2.001E+08 1.959E+07 7.366E+06 4.549E+07 NP62
Mouse 6 4.503E+06 1.786E+07 4.851E+06 9.319E+06 9.400E+06 NP62
Mouse 7 5.514E+06 1.108E+08 4.326E+07 1.511E+07 1.330E+08 NP63
Mouse 8 4.155E+06 6.127E+07 2.146E+07 6.370E+06 1.321E+08 NP63
Mouse 9 5.034E+06 7.958E+07 5.358E+07 1.137E+07 9.709E+07 NP63
Mouse 10 3.517E+06 3.027E+07 1.215E+07 8.739E+06 1.026E+07 NP59
Mouse 11 3.577E+06 3.060E+07 8.146E+06 6.537E+06 6.367E+06 NP59
Mouse 12 4.011E+06 2.507E+07 3.580E+06 6.698E+06 5.715E+06 NP59
Mouse 13 4.149E+06 8.955E+06 5.492E+06 5.795E+06 5.320E+06 Control
Tab.4.2.6 : Radiant efficiency of the organ measurements. This table includesthe collected raw data evaluated in Fi .4.2.6 and Fi .4.2.7.
Fig.4.2.7 : Particle contamination of the skin samples. Emission measured forthe skin samples after the subtraction of the emission determined for the blood
samples. The results for all mice are displayed compared to the results for the
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Fig.4.2.8 : BFI images of the extracted samples. Each image shows the results ofall NPs: (A) liver, (B) skin, (C) lungs, (D) spleen and (E) blood.
5. Discussion
The purpose of our studies was to evaluate the eligibility of a series of surface
coated polystyrene based NPs for a potential medical application in the future.
Before a further assessment of the particle properties and their impact, basic
research in terms of particle toxicity and cellular uptake was required. With this
objective we started a series of test including in vitro as well as in vivo experiments.
In the in vitro studies we employed a model established by Zupke et al. for the
evaluation of particle uptake in DCs and possible cytotoxic effect.[11] Concerning
the in vivo experiments we were mainly interested in the particle distribution to the
organs. Preliminary test performed in our group already showed that there was no
NP
61
62
6359
control
61 62
63 59
control
NP
61
62
6359
control
61
6263
59control
NP61
62
63
59
control
A B
DC
E
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direct toxicity detectable during the first 96h.
There are lots of studies proving that many parameters have an influence on the
cellular uptake of NPs and also on the prevalent mechanism of particle
internalization. Because of that it was important for our work to narrow down the
range of variables that may affect the outcomes of our experiments. Under this
premise we had chosen to test polystyrene particles that were all created in a
miniemulsion process and that were similar in size. The miniemulsion process has
the advantage that it can produce polymeric NPs of a size ranging from 50 - 500
nm, hence very small particles can be produced also with a narrow size
distribution. Additionally it is possible to add functional groups to the particles. This
is of great biological relevance as the functional groups located on the surface of a
particle are the first site to be recognized by a cell. [12]
Our experiments covered two NPs that were not functionalized: The particle GB-
PS 61 which was created using a non-ionic surfactant (Lutensol AT50) and the
particle BR 59 wich was created using an ionic surfactant (SDS). The remaining
two particles were both created with Lutensol AT50 and contained different
functional groups. GB-PS 62 was equipped with a Carboxyl-(COOH) group and
GB-PS 63 was functionalized with an amino- (NH2) group.
Our findings demonstrated that the particles all were taken up well by monocyte-
derived DCs in ourin vitro approach. This result was anticipated as DCs constantly
probe their environment for foreign substance that can be ingested. This confirms
the finding of other groups. Manolova et al. have shown that small particles in the
range of 20-200 nm were taken up by lymph node resident DCs. [13] Zupke et al.
showed that polystyrene nanoparticles without functional groups as well as
particles with amino or carboxy functional groups are readily taken up by DCs
without showing toxic effects. [11] Our experiments did support that there is no
toxicity for the carboxy-functionalized particle GB-PS 62 and for the non-modified
particle BR 59. But still there was a decreased viability determined in correlation
with an increased concentration of the un-functionalized particle GB-PS 61. This
suggests that this particle may be toxic but it may as well have other reasons. On
the one hand a plausible explanation for the decreased viability would be that I
was inexperienced in terms of cell culture, thus the cells were exposed to higher
stress levels. The stress was additionally increased by the treatment with NP
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resulting in a higher apoptosis rates. On the other hand the presence of particle
caused distortions in the 7AAD measurements due to an overlap of the emission
spectra of BODIPY and 7AAD. That may have influenced the outcomes for the
viability determination. For GB-PS 63 the 7AAD distortions did not allow a
significant analysis of the viability. This also may have caused the decrease in thedetermined viability at 300 g/ml BR 59. Probably it is possible to apply an
improved compensation for this distortion. However, the data was insufficient to
draw conclusions on the particle toxicity, further tests are required for GB-PS 61
and GB-PS 63.
Concerning the uptake rate the results were more well-defined. An optimal uptake
for GB-PS 62 was achieved at 150 g/ml without affecting viability. Doubling the
concentration only achieved an increase of the uptake by less than 20 %. For GB-
PS 61 the maximum uptake was reached at 75 g/ml with minor effects on viability.
GB-PS 63 had an optimal uptake at 150 g/ml. Only for BR 59 a significant
improvement of the uptake was achieved at 300 g/ml. As the decrease in viability
was very low this concentration seems optimal for this particle.
The cLSM analysis showed that the maturation of the DCs worked well as
indicated by the typical morphology including the development of dendrites. Some
of the cells were completely stained red (in contrast to the expected staining of the
cell membrane only). This can be explained as in apoptotic or dead cells the
plasma membrane stain could leak inside the cell. All particles had the tendency to
form aggregates. The agglomeration especially of smaller particle has also been
reported by other groups [14]. In our experiment we used particles from two
different charges. Except for NP 59 the other measurements were performed with
particles from the first charge, which were stored at room temperature and were
already several months old. In another experiment we compared the older
particles to a fresh charge of particles in a cLSM approach. The results revealed
that the older particles showed stronger aggregation and had seemed to have a
negative influence on the cellular condition as well. This is underlined by the cLSM
results shown for BR 59 which was also from a fresh charge that was only a few
days old and stored at 4C. For this particle the cells were in a good condition and
the agglomeration was weaker than for the older GB-PS NPs.
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All particles were mainly located within the DCs. Only few particles showed an
attachment to the cell surface as already mentioned in the results (see Fig.4.1.6 B).
However, especially some large aggregates were not ingested or were released
again from dead cells. An interesting effect we discovered was the great variation
of colors in the images obtained especially for NP 61 and NP 63. At higherconcentrations of these particles the overlay view of the fluorescent channels
resulted in colors ranging from light blue to pink. We suspected that these colors
may be the result of an overlap of the different fluorescence signals. Still, not all
the colors could be explained by the superimposition of the fluorescent signals.
Some of the NP 63 and NP 61 returned a white color signal. We suggest that this
could be a quenching effect. The clearance of this phenomenon our group referred
to as candy effect still requires further investigation, especially as the effectinterferes with the proper interpretation of the results.
Another interesting aspect that opened up questions is the location of BR 59
around the nucleus as shown in Fig.4.1.14 and Fig.4.1.15.
The in vivo results showed that the particles showed differences in the
biodistribution. We researched the particle occurrence in the regions of the liver
and lungs as well as particle incidence in the snout region. During other
experiments we tested different particle concentrations and determined an optimal
concentration of 3.7 % (v/v) for the in vivo detection.
After intravenous injection, it is known that that NPs are rapidly detected by the
immune system and cleared from the blood stream via phagocytosis. The particle
is then delivered to the mononuclear phagocyte system (MPS) such as lungs, liver,
spleen and bone marrow. [14] This was confirmed by our findings as well for the
kinetic study as for the following examination of the organs. All particles showed up
mainly in the liver and to a lesser extent in the spleen. Interestingly the non-
modified particle GB-PS 61 and the amino functionalized GB-PS 63 were
detectable in the lung region immediately after the administration where they were
still present after 96h as is was demonstrated in the organ analysis. Both particles
also were present in the skin after 96h. This makes especially NP 63 very
interesting for further research as the location of this particle is not limited to the
liver, the particle also delivers the strongest signal and it has been shown by other
groups that the amino functionalization can improve the particle uptake by different
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6. References
[1] Janeway C.A., et al. Immunobiology - The immune system in Health and
disease, 5th edition, 2001, p.3f,12f
[2] Delves P.& Roitt I., The Immune System First of two parts. New Englandjournal of medicine, 2000
[3] Delves, P. & Roitt I., The Immune System Second of two parts. New En