Bachelorarbeit Stefan Hellberg 2011

Targeting of human dendritic cells with surface optimized 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
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.
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)................................................................................................................ 2 1.3 2. 2.1 Nanoparticles, medical and pharmacological relevance .............................. 3 Materials and Methods .................................................................................... 4 Materials.................................................................................................... 4 Equipment .......................................................................................... 4 Chemicals and consumption objects .................................................. 5 Surgical instruments ........................................................................... 6 Drugs, Media and Additives ................................................................ 7 Zytokines ............................................................................................ 7 Cellculture media ................................................................................ 7 Buffer and Solutions ........................................................................... 8 Antibodies ........................................................................................... 8 Laboratory animals ............................................................................. 9 NSG Mouse haltung/ftterung ......................................................... 9 Nanoparticles .................................................................................. 9 Isolation of peripheral blood lymphocytes ......................................... 10 Generation of dendritic cells (DCs) from blood monocytes ............... 10 Cryo-conservation of cells ................................................................. 11 Flow cytometry................................................................................... 11 Confocal laser scanning microscopy ................................................ 13 In vitro examination of DCs with NP ................................................. 13 Extraction of organs / preparation of in vivo samples ....................... 14 Biofluorecence Imaging (BFI) ........................................................... 14 2.1.1 2.1.2 2.1.3 2.1.4 2.1.5 2.1.6 2.1.7 2.1.8 2.1.9 2.1.10 2.1.11 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6 2.2.7 2.2.8 3. 4.
Methods .................................................................................................. 10
Nomenclature, Abbreviations and Units ........................................................ 15 Results .......................................................................................................... 15 4.1 In Vitro Examination of DCs loaded with NP ........................................... 15 Viability determination of DCs ........................................................... 16 Determination of NP uptake into DCs ............................................... 20 cLSM results for NP GB-PS 62 ......................................................... 21 Results NP 61 ................................................................................... 24 Results NP 63 ................................................................................... 27 Results NP 59 ................................................................................... 28 4.1.1 4.1.2 4.1.3 4.1.4 4.1.5 4.1.6 4.2
Biodistribution of NP in a Mouse model ................................................... 32
4.2.1 4.2.2 5. 6.
Mouse kinetics .................................................................................. 32 Organ distribution ............................................................................. 39
Discussion ..................................................................................................... 42 References .................................................................................................... 47
1. Introduction
1.1 The role of Dendritic cells in the immune system
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 1
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, a change 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
Dendritic cells, role in the development of an acute Graft vs. Host 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 2
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 pharmacological relevance

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 and chemical 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
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 into NOD.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 Autoclave Centrifuge CO2-Incubator 37C, 5% CO2 Confocal laser scanning microscope (CLSM) 510UV Flow cytometer Canto with Diva software Freezer -80C Fume hood bench type Fume Adsorber Ice machine UBE50/35 In Vivo Imaging System (IVIS) Microliter pipettes transfer pipette R S 0,5-10l / 10-100k / 20-200l / 100-1000l Tec Lab (Knigstein) KSG Sterilizers (Olching) Kendro-Heraeus (Langenselbold) Heraeus (Langenselbold) Zeiss (Oberkochen) BD Biosciences (Heidelberg) Heraeus/Kendro (Langenselbold) TAZ 19 Medite (Burgdorf) Ziegra (Isernhagen) Xenogen
Nitrogen-Cryo-bank XLC 1370, Nitrogen tank Taylor-Wharton XL-180 Phase contrast microscope for cell culture Pipette aid PipetBoy Refrigerator and freezer combination 4C / -20C Vortex MS2 Minishaker
MVE Europe (Solingen) Tec Lab (Knigstein) Axiovert 25, Zeiss (Jena) IBS Integra Biosciences (VWR Darmstadt) Privileg (Frth) IKA (Staufen)
2.1.2 Chemicals and consumption objects 7-Aminoactniomycine Beaker (glass) 200,500 and 1000 ml Cell strainer Cell culture flask 250 ml culture flask Counting chamber Fuchs-Rosenthal Cover glasses 24x32 mm Culture plates 24-, 48-, 96-well Culture plates Disposable pipettes, sterile 2, 5, 10, 25 and 50 ml DMSO (Dimethyl sulfoxide) DRAQ5 EDTA (Ethylenediaminetetraacetic acid) Eppendorf tubes 200 l, 500 l, 1500 l FICOLL-paque Freezing-boxes Freezing-tubes Cryotube 1.8 ml Freezing-tube rack FACS-tubes for Flow cytometry BD Falcon FACS-tube rack Falcon tubes 50ml BD Falcon tubes BD Bioscience (Erembodegem, B) Schott (Mainz) BD Falcon Greiner (Nrtingen) Schreck (Hofheim) Menzel (Braunschweig) Greiner (Nrtingen) Greiner (Nrtingen) Merck (Darmstadt) Biostatus (Shepshed, UK) Sigma (Deisenhofen) Eppendorf (Hamburg) GE Healthcare (Freiburg) Nalge Nunc (Wiesbaden) Nunc (Wiesbaden) Roth (Karlsruhe) BD Bioscience (Erembodegem, B) Roth (Karlsruhe) BD Bioscience (Erembodegem, B)
Falcon tube racks Gloves (latex) Insulin syringe 0,3 ml 30 G 8mm Isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1trifluoro-ethane) Paraformaldehyde (PFA) PBS (Phosphate-buffered saline), liquid Petri-dishes 94mm, 25mm Pipette tips 0,5-10 l , 10-200 l, 100-1000 l Sodium chloride (NaCl) Syringes 2 ml single-use syringes Trypan blue 2.1.3 Surgical instruments Clamps Straight Clamp 12.5 cm Cat. Nr. 14009 - 12
BD Bioscience (Erembodegem, B) Semperid (Austria) Becton Dickinson (Heidelberg) Abbott (Wiesbaden) Merck (Darmstadt) Gibco BRL (Karlsruhe) Greiner (Nurtingen) Starlab (Ahrensburg) Carl Roth (Karlsruhe) Braun (Melsungen) Merck (Darmstadt)
Forceps Straight forceps 10 cm Cat. Nr. 11050 - 10 Forceps with curved with slotted 0.8 mm x 0.7 mm tip, 10 cm Cat Nr. 11052 - 10 Straight forceps 12 cm Cat. Nr. 11002 - 12
Scissors Surgical Scissors straight sharp/blunt blade 12 cm Cat. Nr. 14001 - 12 Surgical Scissors straight sharp Cat. Nr. 14002 - 12 All Referring to FST - Fine Science Tools (Heidelberg)
2.1.4 Drugs, Media and Additives Aqua dest. AIM-V Medium Ketamin (50 mg / ml) Heparin (Liquemin) Human albumin Human Serum (HS) from healthy donor blood, heat-inactivated at 50C for 30 min. Mixed from 10 to 20 donors. . Penicillin/Streptomycin B.Braun (Melsungen) Gibco BRL (Karlsruhe) Ratiopharm (Ulm) Roche (Grenzach-Wyhlen) Octapharm (Langenfeld) Blood bank of University hospital (Mainz) Gibco BRL (Karlsruhe)
2.1.5 Zytokines Interleukin 1 (IL-1) Interleukin 4 (IL-4) Interleukin 6 (IL-6) Granulocyte macrophage colony-stimulating factor (GM-CSF) Tumor necrosis factor (TNF- ) Prostaglandin E2 (PGE2) Miltenyi (BergischGladbach) R&D Systems (Wiesbaden) Miltenyi (BergischGladbach) Bayer (Leverkusen) Promokine (Heidelberg) Sigma (Deisenhofen)
2.1.6 Cellculture media DC-Medium Medium A Medium B AIM-V with 1% human serum DC-Medium with GM-CSF 800 U/ml and IL-4 1000 U/ml DC-Medium with GM-CSF 1600 U/ml and IL-4 1000 U/ml DC-Medium + with GM-CSF 800 U/ml and IL-4 500 U/ml AIM-V with Human albumin 8% and Heparin (Liquemin) 10 U/ml
Medium C Freezing medium
2.1.7 Buffer and Solutions FACS buffer storage at 4C Narcosis solution Xylazin (Rompun 2%) Ketamin (50 mg/ml) H2O 0.8 ml 2.0 ml 2.8 ml PBS 0.5% BSA 500ml 250mg
storage at 4C max. 14 days
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 blue (application solution) storage at RT Trypan blue H2O Trypan blue stock solution 150 mM NaCl 2,0 g ad 1 l 75 ml 25 ml
2.1.8 Antibodies Antibodies used for flow cytometry as following Labeling CD45 CD11c CD80 CD83 CD86 CD14 CD19 CD3 Specificity Mouse Mouse Mouse Mouse Mouse Mouse Mouse Mouse Conjugation FITC / PE / APC APC FITC FITC PE PE FITC APC Producer Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter
HLA-DR Mouse PE Beckman Coulter Tab.1: Overview of antibodies used to label surface antigens. The following conjugations were used: Fluoresceinisothiocyanate (FITC), Phycoerythine (PE) and Allophycocyanine (APC)
2.1.9 Laboratory animals In our in vivo 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 17707/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) Particle Functional group Surfactant Diameter, nm (STDN, %)
GB-PS-61 Lutensol AT 50 145 (17.7) (NP 61) GB-PS-62 COOH Lutensol AT 50 139 (14.0) (NP 62) GB-PS-63 NH2 Lutensol AT 50 153 (8.9) (NP 63) BR 59 SDS 125 (15.7) (NP 59) Tab.2: Overview of Nanoparticles used in the experiments. Concentration of NP samples is 3.7% and were provided by the . All NP are triple loaded with a) IR-dye780 b) BODIPY c) Platin(II)acac
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 was
obtained 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 as white 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 10
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 Nitrogen bank 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 a fluorochrome. 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 11
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 7AAD 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 vitro examination 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) were harvested 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 previous cervical 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 intravenously into 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 placed in 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
14
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 Vitro Examination 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
a.
b.
c.
d.
Fig.4.1.1 : Evaluation of the obtained FACS data. The blots show the exemplified results of the APC and 7AAD measurement comparing a negative control sample (a. and b.) to an APC/7AAD stained sample (c.) and (d.). On the left hand 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 represents the intensity of the 7AAD signal while the y-axis represents the according amount of 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 main populations were gated s negative while events outside these gates were regarded as positive events for the according parameter. (c.) and (d.) show the results of a DC sample stained with CD11c and 7AAD. In (c.) the main population is located in quadrant A1 / A2 and hence CD11cpos. These cells were gated as DCs. (d.) The histogram only views the events of the gate DCs. The first peak represents 7AADneg cells (as determined in (b.)) gated as viable. The remaining events are 7AAD positive. The second peak is gated as Apoptotic and the third peak is gated as Dead 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
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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 DCs Viable Apoptotic Dead Total events
Number of events 8036 7392 246 398 10000
% Total 80.36 73.92 2.46 3.98 100.00
% Gated 80.36 91.99 3.06 4.95 100.00
Tab 4.1.1 : Quantification of the events gated in Fig.4.1.1. An overview of the number of events contained by gates presented in Fig.4.1.1 (c.) and (d.) The amount of events can be displayed as a total number of events as well as a fraction of either the total number of events measured (% Total) or as a fraction of the gate that is displayed in a plot (% Gated)
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.
17
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 7AAD neg 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
18
Probes NC (negative control) DC+GB-PS 62 [25 g/ml] DC+GB-PS 62 [75 g/ml] DC+GB-PS 62 [150 g/ml] DC+GB-PS 62 [300 g/ml]
viable 92.22 91.89 92.32 90.58 87.45
apoptotic 3.81 2.39 2.71 2.32 4.34
dead 3.98 5.74 4.95 7.09 8.20
Tab 4.1.2 : Vitality of DCs incubated with NP GB-PS 62. Viable, apoptotic and Dead cells as % Gated of the CD11cpos population. The negative control sample (NC) shows the vitality of DCs without the addition of NP whereas the other samples represent the vitality at increasing levels of NP (25, 75, 150 and 300 g/ml).
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 concentration the viability rates has lost roughly 5 % and Fig.4.1.2 : Vitality of DCs in dependence on GB-PS 62 concentration. This figure visualizes the data of Tab.4.1.2 and shows vital, apoptotic and dead cell percentage in relation to the NP concentration. the dead percentage cells of 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 GBPS 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.
19
4.1.2 Determination of NP uptake into DCs The second aspect to be analyzed in our in 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 NP pos as exemplified in Fig.4.1.3
a.
b.
Fig.4.1.3 : FL1 signals of loaded and unloaded DCs. (a.) shows the FL1 signal of a negative control sample. Events below the intensity peak were gated as NP negative; all events above were gated as NP positive. (b.) shows the signal of DCs that were incubated with 300 g/ml GB-PS 62. 93 % of the events were estimated as NP positive. . 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.
20
Probes NC DC+GB-PS 62 [25 g/ml] DC+GB-PS 62 [75 g/ml] DC+GB-PS 62 [150 g/ml] DC+GB-PS 62 [300 g/ml]
MFI 0.7 5.1 4.9 6.2 6.8
% Gated 0.27 88.03 87.63 88.96 92.41
Tab 4.1.3 : GB-PS 62 MFI and %Gated of the NP positive fraction. The samples contain an increasing amount of NP 62 in addition to a negative control sample (NC).
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 only a single approach, the
following experiments were conducted approach conditions. experiments Fig.4.1.4 : MFI of cells gated as NP . The signal intensity correlates with the particle concentration 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
pos
in
duplicate identical these statistical standard
under For the the
mean
and
deviation of the according results were determined and
21
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. A B C
Fig.4.1.5 : cLSM image of mDCs with incorporated GB-PS 62 [25 g/ml]. The particles contain BODIPY fluorescent dye (green). Cell membrane stain (red) was performed with CellMask Orange. Hoechst 33342 (blue) was used for nuclear staining. The different channels recorded are shown separately in pictures (A) to (D). An overlay of the fluorescence channels is given in (E).
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
22
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. A B C
Fig.4.1.6 : cLSM image series of mature DCs with increasing concentration of incorporated GB-PS 62. (A) negative control; (B) 25 g/ml; (C) 75 g/ml (D) 150 g/ml (E) 300 g/ml. 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.
23
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 NC DC+GB-PS 61 [25 g/ml] DC+GB-PS 61 [75 g/ml] DC+GB-PS 61 [150 g/ml] DC+GB-PS 61 [300 g/ml] viable 90.06 87.20 76.30 65.43 66.91 apoptotic 6.58 7.27 14.07 18.87 23.29 dead 3.33 5.54 9.69 15.74 9.66
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 increasing concentrations of NP61. (Average from duplicated approach, STDN given in Tab.4.1.5)
Sample NC DC+GB-PS 61 [25 g/ml] DC+GB-PS 61 [75 g/ml] DC+GB-PS 61 [150 g/ml] DC+GB-PS 61 [300 g/ml] STDN (%) viable 1.49 3.10 3.39 3.84 8.27 apop. 7.07 32.46 3.41 0.45 12.84 dead 27.63 6.32 21.78 15.60 28.53
Tab 4.1.5 : Standard deviations (%) for the results presented in Tab.4.1.4.
Sample NC DC+GB-PS 61 [25 g/ml] DC+GB-PS 61 [75 g/ml] DC+GB-PS 61 [150 g/ml] DC+GB-PS 61 [300 g/ml]
MFI
+/- STDN
1.7 2.1 2.9 2.5

2.6
0.1 0.1 0.0 0.1

0.1
% Gated NPpos 1.86 16.63 43.87 62.38 76.30
+/- STDN 0.93 3.17 0.00 1.53 0.92
Tab 4.1.6 : GB-PS 61 uptake. Amount of cells gated as GB-PS 61 positive and MFI with the according standard deviations (STDN) for all concentrations.
24
The obtained data was blotted in Fig.4.1.7
Fig.4.1.7 : DC Vitality and NP Uptake in dependence on particle concentration. The Figure shows the amount of viable, apoptotic and dead cells (A) and the estimated rate of uptake (B) at increasing rates of NP concentration compared to a negative control sample (NC).
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 of dead 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.
25
Fig.4.1.8 : cLSM image series of mature DCs with increasing concentration of incorporated GB-PS 61. (A) negative control; (B) 25 g/ml; (C) 75 g/ml (D) 150 g/ml (E) 300 g/ml. 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 is particle clearly
concentration
evident the image series. The NP gives a strong Fig.4.1.9 : Split channel view of DCs containing GBPS 61 [300 g/ml] 26 signal and tends to form
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
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 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 NC DC+GB-PS 63 [25 g/ml] DC+GB-PS 63 [75 g/ml] DC+GB-PS 63 [150 g/ml] DC+GB-PS 63 [300 g/ml]
MFI
+/- STD
1.7 5.5 15.8 18.9 19.2
0.2 2.1 1.1 1.3 0.2
% Gated NPpos 1.31 67.91 90.07 94.70 99.32
+/- STD 1.31 67.91 90.07 94.70 99.32
Tab.4.1.7 : GB-PS 63 uptake. Amount of cells gated as GB-PS 63 positive and MFI with the according standard deviations (STDN) for all concentrations.
27
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 A B C
Fig.4.1.11 : cLSM image series of mature DCs with increasing concentration of incorporated GB-PS 61. (A) negative control; (B) 25 g/ml; (C) 75 g/ml (D) 150 g/ml (E) 300 g/ml. 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.
28
Probes NC DC+ BR 59 [25 g/ml] DC+ BR 59 [75 g/ml] DC+ BR 59 [150 g/ml] DC+ BR 59 [300 g/ml]
viable 80.47 77.78 81.40 80.20 70.77
apoptotic 14.40 16.32 13.04 15.19 24.59
dead 5.06 5.83 5.49 4.43 3.90
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 increasing concentrations of NP BR 59. (Average from duplicated approach, STDN given in Tab.4.1.11)
STDN (%)
viable 0.00 2.70 2.13 1.20 20.72
apoptotic 0.00 21.79 13.96 0.49 51.48
dead 0.00 24.70 0.91 21.90 33.50
Tab.4.1.11 : Standard deviation for the results presented in Tab.4.1.10
MFI 2.30 5.10 21.85 31.25 130.60
STDN 0.20 0.50 6.75 0.85 21.50
% Gated NPpos 0.54 49.75 73.95 86.08 89.69
STDN 0.08 5.08 5.21 0.83 1.36
Tab.4.1.12 : BR 59 uptake into DCs. Amount of cells gated as BR 59 positive and MFI with the according standard deviations (STDN) for all concentrations
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
29
Fig.4.1.12 : DC vitality and uptake of BR 59 in dependence on NP concentration. The Figure shows the amount of viable, apoptotic and dead cells (A) and the estimated rate of particle uptake (B) at increasing rates of NP concentration compared to a negative control sample (NC). relatively big jump can be observed at the highest concentration.
Fig.4.1.14 : cLSM Z-Stack analysis of DCs with BR 59. The image series shows the same location with increasing depths.
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
30
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
Fig.4.1.15 : cLSM image series of mature DCs with increasing concentration of incorporated BR 59. A) negative control; (B) 25 g/ml; (C) 75 g/ml (D) 150 g/ml (E) 300 g/ml. The nuclear staining was performed using DRAQ5
images of two cells with incorporated NP with an increasing depth. In both cells the particle is located around the nuclear region.
31
4.2
Biodistribution 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.
1 2 (NC)
4
A B C
Fig.4.2.1 : Bio distribution of BR 59 at different time points after the injection. 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 and was used as reference. 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) 32
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. A maximum 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 a less 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.
NP 59 Mouse 1 time (h) Snout Lung L Lung R Liver Snout Lung L Lung R Liver Snout Lung L Lung R Liver Snout Lung L Lung R Liver 0 8.310E+06 1.042E+07 8.491E+06 1.784E+07 7.016E+06 7.387E+06 7.610E+06 1.454E+07 6.819E+06 7.400E+06 7.223E+06 1.383E+07 5.917E+06 5.602E+06 5.407E+06 5.795E+06 4 8.490E+06 9.227E+06 9.573E+06 1.489E+07 7.348E+06 7.452E+06 7.451E+06 1.098E+07 8.498E+06 7.381E+06 7.474E+06 1.249E+07 6.012E+06 6.281E+06 6.180E+06 6.642E+06 24 1.075E+07 1.036E+07 9.736E+06 1.244E+07 8.546E+06 8.131E+06 7.583E+06 1.087E+07 8.879E+06 8.455E+06 7.460E+06 1.059E+07 6.687E+06 6.075E+06 6.102E+06 6.742E+06 48 9.429E+06 1.033E+07 8.685E+06 1.228E+07 8.319E+06 7.949E+06 7.864E+06 1.098E+07 8.973E+06 8.205E+06 7.307E+06 1.034E+07 6.389E+06 5.949E+06 6.027E+06 6.563E+06 96 9.290E+06 8.581E+06 8.462E+06 1.069E+07 8.472E+06 7.619E+06 7.498E+06 1.061E+07 8.570E+06 7.607E+06 7.346E+06 1.082E+07 6.168E+06 6.217E+06 6.257E+06 6.860E+06
Mouse 2
Mouse 3
Control
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 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 33
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).
1 2 (NC)
4 A B C
Fig.4.2.2 : Bio distribution of GB-PS 61 at different time points after the injection. 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 and was used as reference.
NP 61 time (h) Mouse 1 Snout Lung L Lung R Liver Mouse 2 Snout Lung L Lung R Liver Mouse 3 Snout Lung L Lung R Liver Control Snout Lung L Lung R Liver 0 1.539E+07 2.752E+07 1.205E+07 1.540E+07 1.721E+07 1.904E+07 1.349E+07 1.244E+07 1.419E+07 1.712E+07 9.801E+06 1.058E+07 6.068E+06 5.565E+06 5.467E+06 5.610E+06 4 2.681E+07 2.364E+07 2.011E+07 3.122E+07 1.488E+07 1.836E+07 1.170E+07 1.625E+07 1.559E+07 1.380E+07 1.232E+07 1.468E+07 6.073E+06 6.115E+06 5.989E+06 6.196E+06 24 2.302E+07 1.885E+07 2.097E+07 2.570E+07 1.448E+07 1.179E+07 1.548E+07 1.630E+07 1.306E+07 1.361E+07 1.335E+07 1.517E+07 6.837E+06 6.677E+06 6.349E+06 6.667E+06 48 2.502E+07 2.655E+07 1.548E+07 2.610E+07 1.325E+07 1.549E+07 1.227E+07 1.693E+07 1.294E+07 1.724E+07 1.102E+07 1.544E+07 6.108E+06 6.022E+06 5.489E+06 6.094E+06 96 2.078E+07 1.930E+07 1.545E+07 2.563E+07 1.246E+07 1.527E+07 1.129E+07 1.712E+07 1.257E+07 1.380E+07 1.054E+07 1.530E+07 6.246E+06 6.289E+06 6.033E+06 6.339E+06
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 for the ROIs defined for Snout, Liver as well as for left and right lung 34
1 2 (NC)
4 A B C
E at different time points after the D Fig.4.2.3 : Bio distribution of GB-PS 62 injection. 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 and was used as reference.
NP 62 Mouse 1 time (h) Snout Lung L Lung R Liver Snout Lung L Lung R Liver Snout Lung L Lung R Liver Snout Lung L Lung R Liver 0 6.864E+06 7.430E+06 6.563E+06 8.482E+06 1.091E+07 1.304E+07 1.179E+07 5.117E+07 6.081E+06 6.259E+06 5.723E+06 6.558E+06 6.051E+06 6.225E+06 5.825E+06 6.127E+06 4 1.014E+07 7.486E+06 8.326E+06 8.787E+06 1.657E+07 1.410E+07 1.137E+07 3.820E+07 9.152E+06 6.753E+06 6.573E+06 7.430E+06 6.598E+06 6.215E+06 6.235E+06 6.526E+06 24 1.658E+07 9.273E+06 9.536E+06 9.733E+06 1.763E+07 1.415E+07 1.264E+07 3.558E+07 1.634E+07 1.063E+07 9.079E+06 9.734E+06 7.102E+06 6.229E+06 6.197E+06 6.916E+06 48 1.353E+07 9.378E+06 9.509E+06 9.237E+06 1.698E+07 1.434E+07 1.289E+07 3.837E+07 9.548E+06 1.016E+07 1.124E+07 1.062E+07 6.732E+06 6.222E+06 6.046E+06 6.300E+06 96 1.089E+07 8.056E+06 8.326E+06 8.677E+06 1.534E+07 1.350E+07 1.297E+07 4.888E+07 1.254E+07 9.755E+06 9.049E+06 1.025E+07 7.121E+06 6.467E+06 6.538E+06 6.769E+06
Mouse 2
Mouse 3
Control
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 for the ROIs defined for Snout, Liver as well as for left and right lung 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
35
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
1 2 (NC)
4 A B C
Fig.4.2.4 : Bio distribution of GB-PS 63 at different time points after the injection. 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 and was used as reference. 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.
36
NP 63 Mouse 1
Mouse 2
Mouse 3
Control
time (h) Snout Lung L Lung R Liver Snout Lung L Lung R Liver Snout Lung L Lung R Liver Snout Lung L Lung R Liver
0 2.458E+07 4.044E+07 2.202E+07 2.385E+07 1.454E+07 2.483E+07 1.260E+07 1.652E+07 2.095E+07 1.768E+07 1.859E+07 1.616E+07 5.791E+06 5.474E+06 5.365E+06 5.800E+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 for the ROIs defined for Snout, Liver as well as for left and right lung 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.
Fig.4.2.5 : Particle distribution analysis, overview of all measurements. Each blot 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) For the further evaluation of the collected data the results were summarized as
37
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 0.500E 7 for all ROIs. At the liver region 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 the radiant 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.
38
NP 61 61 61 62 62 62 63 63 63 59 59 59 Control
Injection Efficiency (%) 100 100 100 50 50 20 50 50 50 100 50 50
Injection sequence 1 2 3 1 2 3 1 2 3 1 2 3 1
Date of birth 05/01/2011 29/12/2010 29/12/2010 05/01/2011 29/12/2010 29/12/2010 29/12/2010 03/11/2010 29/12/2010 29/12/2010 03/11/2010 29/12/2010 29/12/2010
Tab.4.2.5 : Injection efficiency and order. 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 examined organs 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 39
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 59 demonstrated that this particle is
Fig.4.2.6 : Particle distribution to the examined organs after 96h. Each blot shows the results obtained for one particle. All tested mice are displayed compared to a NP negative control (purple).
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.
40
1,600E+04 1,400E+04 1,200E+04
Skin (Blood substracted) after 96h

Mouse 1 Mouse 2 Mouse 3 Control
Emission
1,000E+04 0,800E+04 0,600E+04 0,400E+04 0,200E+04 0,000E+00 NP61 NP62 NP63 Nanoparticle NP59
Fig.4.2.7 : Particle contamination of the skin samples. Emission measured for the 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 negative control (purple)
Subject Mouse 1 Mouse 2 Mouse 3 Mouse 4 Mouse 5 Mouse 6 Mouse 7 Mouse 8 Mouse 9 Mouse 10 Mouse 11 Mouse 12 Mouse 13
Blood Liver Spleen Skin Lung 4.607E+06 8.709E+07 4.227E+07 1.836E+07 9.152E+07 4.047E+06 6.176E+07 3.608E+07 8.232E+06 8.430E+07 3.838E+06 6.324E+07 2.185E+07 7.028E+06 1.547E+08 3.668E+06 1.213E+07 5.632E+06 8.339E+06 7.329E+06 3.785E+06 2.001E+08 1.959E+07 7.366E+06 4.549E+07 4.503E+06 1.786E+07 4.851E+06 9.319E+06 9.400E+06 5.514E+06 1.108E+08 4.326E+07 1.511E+07 1.330E+08 4.155E+06 6.127E+07 2.146E+07 6.370E+06 1.321E+08 5.034E+06 7.958E+07 5.358E+07 1.137E+07 9.709E+07 3.517E+06 3.027E+07 1.215E+07 8.739E+06 1.026E+07 3.577E+06 3.060E+07 8.146E+06 6.537E+06 6.367E+06 4.011E+06 2.507E+07 3.580E+06 6.698E+06 5.715E+06 4.149E+06 8.955E+06 5.492E+06 5.795E+06 5.320E+06
NP NP61 NP61 NP61 NP62 NP62 NP62 NP63 NP63 NP63 NP59 NP59 NP59 Control
Tab.4.2.6 : Radiant efficiency of the organ measurements. This table includes the collected raw data evaluated in Fig.4.2.6 and Fig.4.2.7.
41
NP 61 62 63 59 control
NP 61 62 63 59 control
NP
61 62 63 59 control
NP
61 62 63 59 control
61
62
63 control
59
Fig.4.2.8 : BFI images of the extracted samples. Each image shows the results of all 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 42
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 GBPS 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 monocytederived DCs in our in 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 43
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 the determined 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 GBPS 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. 44
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 higher concentrations 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 effect interferes 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 nonmodified 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 45
cell types. [1] Also the presence of this particle in the skin makes it an interesting object for the possible targeting of Langerhans cells. The finding of NP 63 in the blood after 96h, because as mentioned above, usually NP is cleared from the blood stream very efficiently. This prolonged maintenance of NP 63 in the blood makes it an interesting target for drug delivery. Usually only stealth particles like polyethylene glycol (PEG) modified particles are not detected by macrophages as the most particles quickly fall prey to opsonization tagging them as targets for phagocytes [14] The location of NP in the snout was monitored as well, the finding show that all GB-PS particles were present in this region. The reason for that remains unclear and needs further research. It is possible that the particle arrived through the salivary glands. Another explanation would be that particle was taken up from the exterior by contact with excrements or wound cleaning for example.
46
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 England journal of medicine, 2000 [3] Delves, P. & Roitt I., The Immune System Second of two parts. New England journal of medicine, 2000 [4] Hackstein H. & Thomson A.W., Dendritic cells: emerging pharmacological targets of immunosuppressive drugs, Nature Reviews Immunology 4, 2004) [5] Shlomchik, W. D. et al. 1999. Prevention of Graft Versus Host Disease by Inactivation of Host Antigen-Presenting Cells. Science [6] Amarnath, S. et al. 2010. Regulatory T cells and human myeloid dendritic cells promote tolerance via programmed death ligand-1. PLoS. Biol. 8:e1000302. [7] Collin, M. P. et al. 2006. The fate of human Langerhans cells in hematopoietic stem cell transplantation. J Exp. Med. 203:27. [8] Torchilin, V.P. Drug targeting. 2000. Conference Proceedings, France: Elsevier Science Bv.. [9] The supplementation with the desired substances can be controlled and targetoriented.Haag, R. and F. Kratz, Polymer therapeutics: Concepts and applications. Angewandte Chemie-International Edition, 2006. 45(8): p. 1198-1215.] [10] Hillaireau, H. and P. Couvreur, Nanocarriers' entry into the cell: relevance to drug delivery. Cellular and Molecular Life Sciences, 2009. 66(17): p. 2873-2896. [11] Zupke O. et al., Preservation of dendritic cell function upon labeling with
amino functionalized polymeric nanoparticles, Biomaterials, 2010

[12] Elektronenmikroskopische Studien ber polymere Nanopartikel und ihr Potential frbiomedizinische Anwendungen, Martin Dass, Ulm 2010. [13] Manolova V, Flace A, Bauer M, Schwarz K, Saudan P, Bachmann MF., 47
Nanoparticles target distinct dendritic cell populations according to their size, Eur J Immunol, 2008 [14] VJ Mohanraj, Y Chen. Nanoparticles A Review. Tropical Journal of Pharmaceutical Research, June 2006.
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Bachelorarbeit Stefan Hellberg 2011

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Bachelorarbeit Stefan Hellberg 2011

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Targeting of human dendritic cells with surface optimized Nanoparticles

University of Appllied Sciences Bonn-Rhein-Sieg Department of Natural sciences

for the degree of

Bachelor of Science in Applied Biology

Biodistribution of NP in a Mouse model ................................................... 32

Mouse kinetics .................................................................................. 32 Organ distribution ............................................................................. 39

Discussion ..................................................................................................... 42 References .................................................................................................... 47

1.3 Nanoparticles, medical and pharmacological relevance

2. Materials and Methods

Medium C Freezing medium

storage at 4C max. 14 days

3. Nomenclature, Abbreviations and Units

4.1.1 Viability determination of DCs

Gate DCs Viable Apoptotic Dead Total events

Number of events 8036 7392 246 398 10000

% Total 80.36 73.92 2.46 3.98 100.00

% Gated 80.36 91.99 3.06 4.95 100.00

viable 92.22 91.89 92.32 90.58 87.45

apoptotic 3.81 2.39 2.71 2.32 4.34

dead 3.98 5.74 4.95 7.09 8.20

doubled from ~4 % to ~8 %. Also the level of

MFI 0.7 5.1 4.9 6.2 6.8

% Gated 0.27 88.03 87.63 88.96 92.41

duplicate identical these statistical standard

under For the the

deviation of the according results were determined and

1.7 2.1 2.9 2.5

0.1 0.1 0.0 0.1

% Gated NPpos 1.86 16.63 43.87 62.38 76.30

+/- STDN 0.93 3.17 0.00 1.53 0.92

The obtained data was blotted in Fig.4.1.7

fluorescence signal, the increase of is particle clearly

1.7 5.5 15.8 18.9 19.2

0.2 2.1 1.1 1.3 0.2

% Gated NPpos 1.31 67.91 90.07 94.70 99.32

+/- STD 1.31 67.91 90.07 94.70 99.32

viable 80.47 77.78 81.40 80.20 70.77

apoptotic 14.40 16.32 13.04 15.19 24.59

dead 5.06 5.83 5.49 4.43 3.90

viable 0.00 2.70 2.13 1.20 20.72

apoptotic 0.00 21.79 13.96 0.49 51.48

dead 0.00 24.70 0.91 21.90 33.50

Tab.4.1.11 : Standard deviation for the results presented in Tab.4.1.10

MFI 2.30 5.10 21.85 31.25 130.60

STDN 0.20 0.50 6.75 0.85 21.50

% Gated NPpos 0.54 49.75 73.95 86.08 89.69

STDN 0.08 5.08 5.21 0.83 1.36

Biodistribution of NP in a Mouse model

Injection Efficiency (%) 100 100 100 50 50 20 50 50 50 100 50 50

1,600E+04 1,400E+04 1,200E+04

Skin (Blood substracted) after 96h

amino functionalized polymeric nanoparticles, Biomaterials, 2010

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