Bachelorarbeit Stefan Hellberg 2011

7/27/2019 Bachelorarbeit Stefan Hellberg 2011

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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


CD86 Mouse PE Beckman Coulter

CD14 Mouse PE 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


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


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


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


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


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


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.


NP 62 time (h) 0 4 24 48 96


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


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


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


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


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


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


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


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

Documents

Bachelorarbeit Stefan Hellberg 2011