Interaction of silver nanoparticles with human
and porcine skin
vorgelegt von
Diplom-Ingenieur
Sebastian Ahlberg
geb. 20.02.1986, Güstrow, Deutschland von der Fakultät III
Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades
Doktor der Ingenieurwissenschaften Dr.Ing.
-genehmigte Dissertation
Promotionsausschuss:
Vorsitzender: Prof. Dr. Peter Neubauer 1. Gutachter: Prof. Dr. Jens Kurreck 2. Gutachter: Prof. Dr. Roland Lauster 3. Gutachter: PD Dr. Martina C. Meinke
Tag der wissenschaftlichen Aussprache: 10.12.2015
Berlin 2016 D 83
The current work was part of the priority program 1313 "Biological Responses to Nanoscale Particles" of the Deutsche Forschungsgemeinschaft (DFG) at the Department of Dermatology, Venerology and Allergology at the Charité - Universitätsmedizin Berlin from March 2012 until August 2015. It was in collaboration of the Center of Experimental and Applied Cutaneous Physiology (CCP) and the Clinical Research Center for Hair and Skin Science (CRC) which was advised by Prof. Dr. Dr. Jürgen Lademann, PD Dr. Martina Meinke, PD Dr. Annika Vogt and Dr. Fiorenza Rancan.
The work was supervised by Prof. Dr. Jens Kurreck, Institute of Biotechnology, Technische Universität Berlin.
Contents
List of Figures IX
List of Tables XI
List of Abbreviations XIII
1 Introduction 1
1.1 The skin . . . 1
1.2 Human skin cells . . . 3
1.2.1 Keratinocytes . . . 4 1.2.2 HaCaT cells . . . 4 1.3 Nanoparticles . . . 5 1.3.1 Silver nanoparticles . . . 6 1.3.2 Gold nanoparticles . . . 7 1.4 Imaging of nanoparticles . . . 7
1.4.1 Transmission electron microscopy . . . 8
1.4.2 X-ray microscopy . . . 8
1.5 Depth profile of NP uptake . . . 9
1.5.1 2-photon microscopy . . . 9
1.5.2 Raman spectroscopy . . . 10
1.6 Biological response to tissue damage (Cytokine release) . . . 11
1.7 Radical formation . . . 12
1.8 Antioxidant system . . . 12
1.9 Detection of oxidative stress . . . 14
1.10 Aim of this work . . . 17
2 Methods and materials 21 2.1 Materials . . . 21
2.2 Nanoparticles . . . 24
2.2.1 Synthesis of silver nanoparticles . . . 24
2.2.2 Synthesis of gold nanoparticles . . . 24
2.3.3 2PM-induced Fluorescence lifetime imaging microscopy . . . 28
2.3.4 Confocal Raman microscopy . . . 28
2.3.5 Surface enhanced Raman scattering . . . 29
2.3.6 Cryo sections . . . 29
2.3.7 Transmission electron microscopy . . . 29
2.3.8 Scanning transmission X-ray microscopy . . . 30
2.4 Toxicity of nanoparticles towards HaCaT cells . . . 32
2.4.1 Viability assay . . . 32
2.4.2 Cytokine assay . . . 32
2.5 Radical formation . . . 33
2.5.1 Dichlorofluorescein-acetate assay . . . 33
2.5.2 Electron paramagnetic resonance spectroscopy . . . 35
2.5.3 Glutathione assays . . . 36
2.6 UV-irradiation . . . 37
2.7 Statistics . . . 37
3 Results 39 3.1 Detection of nanoparticles within skin and skin cells . . . 39
3.1.1 Porcine skin . . . 39
3.1.2 Human skin . . . 43
3.1.3 Human cells . . . 46
3.2 Toxicity of nanoparticles . . . 53
3.2.1 Cell viability . . . 53
3.2.2 Release of pro-inflammatory cytokines . . . 54
3.3 Radical formation . . . 59
3.3.1 HaCaT cells . . . 59
3.3.2 Antioxidant system . . . 65
4 Discussion 69 4.1 Uptake of dermatological relevant particles . . . 69
4.2 Toxicity of silver nanoparticles . . . 72
4.3 Oxidative stress induced by silver nanoparticles . . . 75
5 Conclusion 81
A Additional data 101
A.1 Fluorescence lifetime imaging microscopy . . . 101
A.2 Confocal Raman microscopy . . . 101
A.3 Shape-dependent uptake of NP . . . 103
A.4 Multi ELISAssay . . . 103
A.5 Caspase-1 . . . 104
B Publications 107
List of Figures
1.1 Scheme of the human skin . . . 2
1.2 Histological section of the skin . . . 2
1.3 Adherent HaCaT cells . . . 5
1.4 Possible uptake mechanisms of NP into the skin . . . 6
1.5 TEM image of AgNP . . . 7
1.6 Principle of the 2-photon and Raman effect . . . 10
1.7 Scheme of ROS reactions in a cell . . . 13
1.8 Basic concept of the EPR spectroscopy . . . 15
1.9 EPR-spectrum of TEMPO . . . 15
1.10 Work plan . . . 19
2.1 TEM image of AuNP rods . . . 25
2.2 Humid chamber . . . 27
2.3 Scheme of the X-ray microscope . . . 31
2.4 Scheme of an ELISA assay . . . 34
2.5 Scheme of the H2DCF-DA reduction . . . 34
2.6 Example for an EPR-measurement . . . 36
2.7 Structural formula of TEMPO and PCA . . . 37
3.1 Average lifetimes of pure AgNP, SC, SS and AgNP-treated porcine skin . . . 40
3.2 Depth profile of AgNP on porcine skin by means of FLIM . . . 40
3.3 Raman spectrum of AgNP-treated porcine skin . . . 41
3.4 Depth profile of AgNP on porcine skin by means of Raman . . . 42
3.5 SERS-signal in AgNP-treated porcine skin . . . 43
3.6 Human skin explants by means of STXM . . . 44
3.7 Uptake of AuNP spheres on human skin explants by means of STXM . . . 44
3.8 Uptake of AuNP rods on human skin explants by means of STXM . . . 45
3.9 Uptake of AgNP on human skin explants by means of TEM . . . 46
3.10 Uptake of AgNP by HaCaT cells by means of STXM . . . 47
3.11 Uptake of AgNP by hMSC by means of STXM . . . 48
3.15 Cell viability after 2 h incubation with AgNP (batch 1) . . . 53
3.16 Cell viability after 24 h incubation with AgNP (batch 1) . . . 54
3.17 Cell viability after 1 h incubation with AgNP (batch 2) . . . 55
3.18 Cell viability after 24 h incubation with AgNP (batch 2) . . . 55
3.19 Interleukin-1α production in HaCaT cells after AgNP-incubation . . . 56
3.20 Interleukin-6 production in HaCaT cells after AgNP-incubation . . . 57
3.21 Interleukin-8 production in HaCaT cells after AgNP-incubation . . . 58
3.22 Radical formation of PCA and TEMPO on HaCaT cells . . . 59
3.23 EPR-signal of AgNP-incubated (batch 1) HaCaT cells . . . 60
3.24 Radical formation of an 1 h AgNP-incubated (batch 1) HaCaT cells . . . 61
3.25 Radical formation of an 1 h AgNP-incubated (batch 2) HaCaT cells . . . 61
3.26 Radical formation of 24 h AgNP-incubated (batch 2) HaCaT cells . . . 63
3.27 Radical formation dependent on the AgNP-concentration of the stock solution . . . 63
3.28 Time-dependent radical formation . . . 64
3.29 Radical formation of AgNP without cells . . . 64
3.30 Radical formation of AgNP-incubated (batch 2) HaCaT cells with the DCF assay . 65 3.31 Total glutathione content of AgNP-incubated HaCaT cells . . . 66
3.32 Reduced and oxidized glutathione content of AgNP-incubated HaCaT cells . . . . 67
3.33 Ratio of reduced and oxidized glutathione . . . 67
4.1 Scheme of the impact of AgNP on the antioxidative system . . . 79
A.1 FLIM image of the skin surface . . . 101
A.2 FLIM image of the epidermis . . . 102
A.3 Raman spectra of AgNP and PVP . . . 102
A.4 Statistic of cellular uptake of AgNP under oxygen and argon . . . 103
A.5 Statistic of cellular uptake of AgNP in different shapes . . . 104
A.6 Cytokine release of AgNP-incubated HaCaT cells . . . 105
List of Tables
2.1 Devices and technical support . . . 21
2.2 Media, Solvent and chemicals . . . 22
2.3 Consumables . . . 23
2.4 Properties of the used nanoparticles . . . 24
2.5 Investigations on the uptake of nanoparticles . . . 25
2.6 Cytokines . . . 33
2.7 Used ELISA . . . 33
2.8 Settings for the EPR spectroscopy with spin labels . . . 36
List of Abbreviations
τ Average lifetime
2PM 2-photon microscopy
a Fluorescence amplitude
AAS Atomic absorption spectroscopy
AF Autofluorescence
AgNO3 Silver nitrate
AgNP Silver nanoparticles
AOS Antioxidant system
Ar Argon
AuNP Gold nanoparticles
CCD Charged coupled device
CRM Confocal Raman microscopy
CTAB Cetrimonium bromide
DCF Dichlorofluorescein-acetate
DNA Desoxyribonucleic acid
E Enzyme
EDTA Ethylenediaminetetraacetic acid
ELISA Enzyme linked immunosorbent assay
EPR Electron paramagnetic resonance
FCS Fetal calf serum
FLIM Fluorescence life time imaging microscopy
GCLC Glutamate-cysteine ligase catalytic subunit
GEE Generalized estimating equation
GM-CSF Granulocyte-macrophage colony-stimulating factor
GPX Glutathione peroxidase GSH Glutathione (reduced) GSHR Glutathione reductase GSS Glutathione synthetase GSSG Glutathione disulfide H2O2 Hydrogen peroxide
HRP Horseradish peroxidase
I Intensity
IFN Interferon
IL Interleukin
MAPK Mitogen activated protein kinase
MED Minimal erythema dose
ML Micro lesion
MRI Magnetic resonance imaging
NADPHX Nicotinamide adenine dinucleotide phosphate oxidase
NF-κB Nucler factor κ B
NP Nanoparticles
O2 Oxygen
P Product
PBS Phosphate buffered saline
PCA 3-Carboxyl-PROXYL
PFA Paraformaldehyde
PMT Photomultiplier tube
PSS Poly(sodium 4-styrenesulfonate)
PVP Polyvinylpyrrolidone
ROS Reactive oxygen species
RPMI Roswell Park Memorial Institute medium
S Substrate
SB Stratum basale
SC Stratum corneum
SEM Standard error of mean
SERS Surface enhanced Raman scattering
SG Stratum granulosum
SHG Second harmonic generation
SLS Swiss Light Source
SM Scanning electron microscopy
SOD Superoxide dismutase
SS Stratum spinosum
STAT Signal transducer and activator of transcription
STXM Scannning transmission X-ray microscopy
T-X Triton-X
TEMPO 2,2,6,6-Tetramethyl-1-piperidinyloxyl
tGSH Total glutathione
TNF Tumor necrosis factor
UV Ultraviolet
Abstract
Brief summary in English
The rising number of antibiotic-resistant bacteria strains point to the need of new strategies dealing with this threat. One mayor outcome in modern biomedical engineering is the development of nano-sized silver particles (AgNP). With its high antibiotic potential AgNP became one of the fastest growing products in nanotechnology. The particles can be found, e.g., in coatings for all kinds of surgical instruments or implants, in textiles, room sprays, creams and in wound dressings. Regarding the prevention and treatment of bacterial infections the negative effect on mammalian cells cannot be ignored. This is way this dissertation will focus its current research on the matter.
The present study addresses the penetration of 70 nm-sized AgNP into the skin, the impact on skin cell viability and the induction of free radicals (ROS). The penetration of these non-fluorescent particles was explored by means of Raman microscopy and fluorescence lifetime imaging microscopy on porcine skin and transmission electron microscopy (TEM) as well as scanning transmission X-ray microscopy (STXM) on human excised skin. The experiments showed no uptake of the AgNP into the viable epidermis in intact skin. In terms of injured skin (e.g., wounds or burned skin) AgNP penetrate into the living tissue and come in contact with the cells, which is shown by TEM and STXM, and are taken-up and stored by the skin cells (HaCaT). The incubation with silver led to morphological changes of the nuclei and the cell membranes, which indicates cell death.
Furthermore it was demonstrated that the presence of AgNP led to a decreasing cell viability and the production of inflammatory cytokines, i.a. IL-1α, IL-6 and IL-8. It was also shown by another method of AgNP-synthesis that the major impact to the cell viability is due to the presence of silver ions (Ag+). Particles produced and stored under air (with a high Ag+ions content) were compared to those synthesized and stored under the inert gas argon (Ar) with a low Ag+ions concentration. It was found that Ag+ions released during particle storage are responsible for most of the AgNP related cytotoxic effects.
This was underlined by the detection of intracellular ROS induced in HaCaT cells incubated with the two AgNP types. ROS formation was investigated by means of electron paramagnetic resonance (EPR) spectroscopy. These experiments explored the high ROS induction by AgNP under oxygen in contrary to the particles under Ar. These results were also confirmed by investigations on one part of the antioxidant system i.e. measurement of the intracellular glutathione.
In conclusion, the present results indicate no dermal uptake of AgNP. In the specific cases of injured skin the AgNP interact with the living cells and cause toxic effects, which might lead to irritation and inflammation. This is associated with an increasing ROS production and therefore the damage on cellular membranes, DNA or proteins. The use of AgNP (Ar) avoid the presence of released Ag+ ions and allow exploiting the beneficial properties of NP i.e. slow and sustained release of Ag+ions. These results highlight the complexity of silver toxicity in medical applications, which need to be thoroughly evaluated for safe use of these nanomaterials in dermatology and other fields.
Abstract
Deutsche Kurzzusammenfassung
Nanopartikel (NP) spielen eine immer größere Rolle im täglichen Leben. Sie besitzen spezielle Eigenschaften, die auf ihren vergrößerten Oberflächen-zu-Volumen-Verhältnis beruhen und sind z.B. besonders reaktiv. In dieser Arbeit werden NP auf ihre Interaktion mit Haut und Hautzellen untersucht. Vor allem Silbernanopartikel (AgNP), die sich auf Grund ihrer antibakteriellen Eigen-schaften steigendem Interesse erfreuen, sind hier in den Fokus gerückt. Speziell in Beschichtungen für OP-Bestecke oder Implantaten, in Textilien, Raumsprays oder Wunddressings sind sie ein fester Bestandteil. Die hohe antibakterielle Wirkung ist eindeutig vorhanden, jedoch können auch Beein-trächtigungen von eukaryotischen Zellen nicht ausgeschlossen werden.
Diese Arbeit untersucht die Aufnahme von NP in die Haut, die Wechselwirkung mit Hautzellen und Induktion von oxidativem Stress. Die Untersuchung der Penetration von ungelabelten Partikeln ist kompliziert und wurde hier durch den Einsatz von Raman Mikroskopie, Fluoreszenzlebens-dauer Mikroskopie, Röntgenmikroskopie und Elektronenmikroskopie an exzidierter Humanhaut und Schweinehaut realisiert. Solange eine intakte Hautbarriere vorhanden war, konnte keine Penetration in der lebenden Hautschichten beobachtet werden. Jedoch treten Schädigungen der Hautbarriere häufig auf (z.B. durch Wunden, Verbrennungen, Operationen), was wiederum die Penetration der NP ermöglicht. So kommt es zum Kontakt zwischen AgNP und Zellen. Diese Situationen wurden simuliert und die Aufnahme in die Zellen konnte nachgewiesen werden. Dies führte zu morpholo-gischen Änderungen der Zelle, wie z.B. der Auflösung des Zellkerns.
Die Toxizität der AgNP auf Keratinozyten wurde untersucht und dabei sowohl eine AgNP-abhängige Verringerung der Zellviabilität als auch die Ausschüttung von inflammatorischen Mediatoren de-tektiert. Durch eine spezielle Synthesemethoden wurde AgNP bereitgestellt, welche eine hohe als auch eine niedrige Anzahl von Silberionen (Ag+) aufwiesen. Es wurde gezeigt, dass die Ag+Ionen größtenteils die Verantwortung für die Toxizität tragen.
Diese Toxizität konnte mittels der Elektronenspinresonanz-Spektroskopie auf oxidativen Stress zurückgeführt werden. Durch die Ag+Ionen werden zum einen Membranen beschädigt und somit Radikale induziert, zum anderen inhibieren diese Ionen das antioxidative System der Zellen, welches für die oxidative Balance sorgt. Daraus entwickelt sich ein Überschuss an freien Radikalen, welcher letztendlich zum Zelltod führt.
Zusammenfassend dokumentiert diese Arbeit, dass die hier untersuchten NP eine intakte Hautbar-riere nicht überwinden können. Hautschädigungen führen jedoch zu einem Durchbruch der NP und zur Interaktion mit Hautzellen. Infolge verschiedener Kaskaden und Reaktionen wird das oxidative Gleichgewicht der Zellen gestört was zum Zelltod führt. Diese Effekte treten bei AgNP, die unter Argon (Ar) synthetisiert wurden verringert auf. Die Ergebnisse dieser Arbeit belegt die Toxizität von AgNP aber auch mögliche Lösungen. Deshalb sollte die Forschung weitergeführt werden, um den Nutzen zu erhöhen und Risiken einzuschränken.
1 Introduction
1.1 The skin
The skin is as outer barrier the most important protection system of the human body towards exogenous threats such as pathogens, cosmic irradiation or natural nanoparticles (NP) i.e. fine dust. Several reviews and books have been published dealing with skin per se and its barrier function [95, 44, 55, 89, 119]. It forms a well-defined defense barrier with its challenging structure and the huge amount of immune cells . Additional functions of the skin, i.a., are:
• Protection against mechanic injuries • Regulation of the body temperature • Barrier against water-loss
• Sensoring organ (e.g., temperature, pressure and pain)
The skin consists of different structures and parts as can be seen in figure 1.1. It is subdivided in three layers - epidermis, dermis and subcutaneous tissue. Structures of importance are the hair follicles, blood vessels and sweat glands.
Epidermis
The epidermis is the upper layer of the skin and consists primarily of keratinocytes which can transform to nuclei-less corneocytes over time. There are several layers distinguished in the epidermis as shown in figure 1.2:
• Stratum corneum (SC) • Stratum granulosum (SG) • Stratum spinosum (SS) • Stratum basale (SB)
Figure 1.1: Scheme of the human skin: Epidermis, dermis, subcutaneous tissue and important structures are shown.1
Figure 1.2: Histological section of the skin: Section was stained with hematoxylin and eosin (HE). Nuclei are stained blue and the cell plasma is red. The different layers of the epidermis are visible.
1.2 Human skin cells The keratinocytes of the basal layer proliferate and differentiate on their way to the upper layer. They are changed from roundly shaped, smaller cells to large, flat cells, finally becoming dehydrated, dead corneocytes, which are forming the top layer of the epidermis the SC and are continuously renewed by desquamation2. This layers’ structure resembles bricks and mortar hence forming a stable ending of the skin. The layers’ forming corneocytes are approx. 1 µm thick and 50 µm in diameter. 10 to 20 corneocyte layers are forming the SC [62]. Additionally, other cells are present in the skin such as Langerhans cells, Merkel cells or melanocytes.
Dermis
The dermis has a less cellular structure and contains blood and lymphatic vessels in comparison to the epidermis. Fibroblasts are the abundant cell type present here. The dermis provides the stability of the skin. Therefore it includes a bunch of elastic fibers such as elastin and tear-resistant fibers made of collagen as example [158]. But this compound of fibers loses its characteristics over time. Also natural and artificial influences can result into damage of dermis components (wich is the main cause of wrinkles). Such influences can be, i.a., sun exposition, smoking, alcohol or stress [80, 43]. The dermis is also a reservoir for cells of the immune system. They rest in the stratum papillare and can migrate into the epidermis when needed [119].
Subcutaneous tissue
This layer of the skin is the connection between the skin itself and lower body parts such as muscles or bones. It is a loose system of connective tissue and subcutaneous fat, which acts as buffering, energy depot and thermoregulator [158].
Skin appendages
These appendages are specialized structures, which develop from epithelial cells of the epidermis. They are independent by their function and morphology but very closely associated to the skin. Dominant appendages in human skin are pilocebaceous unit3, nails or sweat glands.
1.2 Human skin cells
The skin consists of different cell types with different functions. First of all, there are the ker-atinocytes which are responsible for the structure and the barrier function of the skin. Forming a brick and mortar structure, they represent the outer layer of the skin, where the corneocytes (Bricks) are embedded in a lipid matrix (Mortar) [38]. Other cell types are the fibroblasts. These cells
1www.cancer.gov 2
Loss of the upper, dead, horny skin layer.
3
produce collagen and play a big role in the synthesis of the extracellular matrix and wound repair after an injury [44]. Other skin cells act as protection system. The melanocytes are the natural defense against UV (Ultraviolet) radiation. They are resting in the hair follicles and the epidermis [29]. Langerhans cells are part of the immune system and are also localized in the skin [44].
1.2.1 Keratinocytes
As the major cell type in epidermis, keratinocytes are of great interest. The keratinocytes develop in the SB from epidermal stem cells. The process of formation and traveling to the skin surface takes 40 - 56 d in humans [57]. Keratinocytes play a large role in the defense system of the body. They are the first cells coming in contact with pathogens and therefore they produce cytokines and chemokines to recruit the immune system cells [7]. The more they grow towards skin surface, the more connected and protective they get [89]. When they reach the surface they loose their nucleus and organelles and differentiate to corneocytes. These corneocytes are cells which are highly connected by intercellular protein structures, the desmosomes. This barrier is evolutionary adapted to warding off pathogens or natural nanoparticles, i.e., bacteria or fine dust and thereby hard to cross. That is why the keratinocytes are important when it comes to questions about the dermal toxicity and tolerance of e.g., new products.
1.2.2 HaCaT cells
The Human adult low Calcium high Temperature keratinocytes (HaCaT) cell line (Deutsches Krebsforschungszentrum, Heidelberg, Germany) is widely used as a model cell line for investigating the behavior of human skin cells [127]. This immortalized, non-tumorigenic cell line (see fig. 1.3) was cultured from a primary cell culture of human skin keratinocytes. This cell line was developed and described by Boukamp et al. [18]. Full thickness skin was taken from a periphery of a melanoma of a 62 year-old man. The fat and most of the dermis was removed and the skin was placed in trypsin solution. After separating the dermis and epidermis and isolating the cells, both fractions were suspended in medium and finally seeded in petri dish. Cells were cultured and split frequently. The cells showed an immortalization (> 140 passages) but did not became tumorigenic. The HaCaT cells are used as a model for primary keratinocytes. When comparing the two cell types it was shown that both grow as monolayers with comparable morphology and cell adhesion but may show differences in the impact of cytotoxic agents [19]. Also an analogous lipid metabolism (at confluencies < 100 %) is present [128]. HaCaT cells were also widely used for investigations on UV irradiation [133, 78]. Particle toxicity towards HaCaT cells was researched by a lot of groups on titanium dioxide [157], silica particles [118] or on silver nanoparticles [160]. Hence, HaCaT cells become established as a model cell line for keratinocytes.
1.3 Nanoparticles
Figure 1.3: HaCaT cells: Light microscopic image of HaCaT cells grown adherently on a cell culture flask. The cells are grown in their typical squamous shape.
1.3 Nanoparticles
NP are defined to be particular substances in the range between 1 to 100 nm [138]. Manufactured NP play a role in almost every aspect in our environment and also natural NP are present all along. Their origin caused by volcanic activity, dust, bush burnings, smog and many more. These particles are basically based on carbon. The other possible source of NP is human. NP are widely produced for endless reasons because they offer many special properties. Whether in material science, electronics or medicine, NP can be found in every field of life [56, 150, 36, 106, 101]. Special properties of NP are, for example:
• Higher reactivity due to the bigger surface to volume ratio [125] • Special optical properties (Plasmon resonance) [152]
• Ability to reach specific body regions (Hair follicles, blood-brain-barrier) [111]
The high reactivity may lead to problems for the humans, animals and the environment at all. As NP accumulate in waters or in the food chain the risks associated to those particles are not to be underestimated. Investigations showed therefore results in which high pathogenic effects can be seen [39]. One relevant question deals with the possible pathways through the SC into the living skin layers. Those ways are sketched in figure 1.4. But nevertheless there has been no publication to the authors’ knowledge, which can clearly prove the uptake of nano-scaled particles (≥ 40 nm) by the living skin unless the skin is damaged (e.g., by tape stripping, sun burn, pricking) or manipulated (Franz cell) for any reason.
Figure 1.4: Possible uptake mechanisms of NP through the skin barrier into the living skin layers: The SC can be seen as bricks and mortar, with the corneocytes (red) as bricks and the interlipids as mortar (grey). The possible pathways through the SC are a) transcellular, b) intercellular and c) via the HF. Edited from Hadgraft and Lane [55].
1.3.1 Silver nanoparticles
Silver nanoparticles (AgNP) have many applications in biomedical engineering. They are state of the art in many medical uses. Especially the antibacterial potential of AgNP makes them to one important material for coatings or sterile wound dressings [24, 48]. In 2012 Piccinno et al. estimated a production of 55 t / year and showed that about 50 % are used for clothing and almost the other half for medical applications and cosmetics [114]. Several investigations demonstrated that the Ag+ions are responsible for the antibacterial properties of AgNP [26]. The AgNP release Ag+ions slowly and in a sustained manner. This can improve the outcome of antibacterial treatments and wound care management.
Although the high antibacterial potential of AgNP is known, the side effects of this nano-sized material cannot be denied and are not thoroughly investigated yet. Some studies have shown a particle-specific toxicity as well as Ag+ion toxicity [42]. Other studies indicated that the release on Ag+ions due to the presence of oxygen causes the main cytotoxic effects [70]. Necrotic and apoptotic effects such as dysfunctions in the cell cycle, desoxyribonucleic acid (DNA) damages, denaturation of proteins or pertubation of the cell membrane are said to be due to Ag+ions whether on eukaryotic cells such as fibroblasts or bacteria [15, 11, 23]. Silver interacts with biological molecules and forms several complexes as silver chloride or Ag-protein products. It is also inactivated rapidly, what makes a multiple application of active Ag+ions necessary (up to 12 times a day) to guarantee a constant Ag+ion concentration [77]. Hence, efficient silver releasing formulations provide a silver concentration of 70 to 100 ppm, what is sufficient for antibacterial treatment. For example, minimal inhibitory concentrations levels of 8 to 80 µg/mL or 8 to 70 µg/mL were reported for Staphylococcus aureusand Pseudomonas aeruginosa, respectively [28].
1.4 Imaging of nanoparticles
Figure 1.5: AgNP: A TEM image of AgNP (70 ± 20 nm) coated with PVP [26].
1.3.2 Gold nanoparticles
AuNP are an upcoming agent in nanotechnology. They play a role in several applications in chem-istry. Daniel et al. reviewed that AuNP are used as e.g., sugar sensor, catalyzer for, i.a., methanol oxidation or CO2hydrogenation [34].
AuNP are also used in biotechnology as labeling material for X-ray microscopy or electron mi-croscopy. Based on their high density AuNP provide good contrast towards the biological material. They can be found also as delivery agent (Gene gun, uptake by cells). AuNP can also absorb light and emit the absorbed energy in form of heat. For this reason AuNP are prominent as heat source for treating cancer (Hyperthermia) or in drug delivery systems (Opening of bonds or containers). Furthermore these particles can act, as example, as a sensor for surface enhanced Raman scattering (SERS) or quench fluorescence [136].
Therefore, the uptake and of course the toxicity of this NP is of interest. Connor et al. investigated AuNP-spheres (4, 12 and 18 nm) coated with biotin, citrate and cetrimonium bromide (CTAB) [31]. Here, the uptake by human K562 leukemia cells was shown. They reported no particle-induced toxic effects but a reduction of the cell viability caused by AuCl4and unbound CTAB . A review by
Alkilany et al. showed that many investigations demonstrated the non-toxicity of AuNP, whereas other investigations presented contrary results [5]. The AuNP instead of the AgNP keep their shape and size and do not dilute over time [88].
1.4 Imaging of nanoparticles
Today the imaging of NP is a challenging task. There are several ways to detect NP in biological material but every method has its advantages and drawbacks. One method is to label the particles with a fluorescence dye. This offers the advantage that a common fluorescence microscope can picture the labeling. But on the other hand it just pictures the fluorescence of the dye. If the dye is loaded on the particle it could be released during the incubation or investigation. Another
important limit of the light microscopy is the low resolution. Light microscopes have resolution limits which are approx. half of the excitation wavelength4. It could also be possible that the particle itself disaggregates and so one just images a fragment of the particle. Also the size and the chemical properties of the particle are modified when being loaded or labeled with a dye. Thus pure, unloaded/unlabeld NP are very interesting as the particles investigated in this study (AgNP, AuNP) are not fluorescent and hence other light-independent ways are needed.
1.4.1 Transmission electron microscopy
This non-fluorescent and high resolution technique is a widely used method to investigate structures in the submicroscopic scale. In the 1930’s Ernst Ruska invented the transmission electron microscopy (TEM) as a standard imaging system in science and was awarded the Nobel prize for his invention [155]. For this technique the samples are dehydrated and cut into ultrathin slices (70 nm). A beam of electrons produced by a cathode is directed on the sample. The transmitted electrons are visualized for the operator as an enlarged image on a fluorescent phosphor screen and detected on a CCD-chip
5or a photo film. The intensity of the transmitted beam is due to the molecular composition of the
investigated sample. Molecules with a high atomic number show less transmission for the beam than molecules with a low atomic number. Therefore, this method is a way to detect metallic materials in biological specimens caused by the high contrast. Also, TEM provides a very high resolution (approx. 10 nm) which can visualize cellular subunits [112].
1.4.2 X-ray microscopy
A new microscopic technique is the X-ray microscopy. Common techniques and also X-ray microscopy can include preparations steps such as dehydration or freeze drying. Here, the particles can be washed out or just fall off the sample when being sliced. During the preparation of the samples for TEM, e.g., water is removed from the probes by alcohol and other chemicals. During these steps the particles can be removed from the sample and therefore are not detectable. One way to solve these problems are new preparation methods for the scanning transmission X-ray microscopy (STXM) . This technique can analyze native samples. The common way to obtain X-ray irradiation with high intensity is the use of synchrotrons. Full-Field X-ray microscopy for laboratory usage is in research right now [131]. Here, soft X-ray radiation is emitted by a plasma source. The drawbacks with this devices are the low operation time, the radiation intensity, the X-ray irradiation is only slightly or not adjustable at all and spectroscopy is not possible. By means of ring accelerators, electrons are accelerated and transferred to a storage ring where the electrons are deflected and decelerated providing energy in the form of radiation. The soft X-ray irradiation is focused to an endstation, where a sample is scanned to image the absorption of the X-ray irradiation
4
This is due to Abbe’s law.
5
1.5 Depth profile of NP uptake in the sample, which is dependent on the sample material and the electron energy. A resolution of approx. 40 nm can be reached. Also spectroscopic investigations can be done [115, 154].
1.5 Depth profile of NP uptake
A major question is whether the NP pass the skin barrier and reach the living tissue. Established methods such as TEM have been used for such investigations. However, it cannot be eliminated that during the preparation of the samples, the particles are washed out from the tissue. Therefore, other methods are needed to detect the penetration behavior of the particles without the treatment of the samples. Two possible ways are the confocal Raman microscopy (CRM) and the 2-photon microscopy (2PM).
1.5.1 2-photon microscopy
The 2PM technique belongs to the confocal microscopy and uses high-frequently femtosecond lasers to induce nonlinear optical effects, which result in images with high resolutions. Contrary to the ordinary fluorescence microscopy in which one photon excites a molecule, for 2PM two photons are nearly simultaneously inducing the fluorescence (see a) in figure 1.6). Due to this effect (2PM absorption) the laser energy is just the half of the energy used in fluorescence microscopy and therefore less invasive towards the sample or the patient. The less energetic photons are also able to penetrate deeper into the tissue compared to higher energetic photons, which allows for information from lower skin layers. Also the excitation of the fluorescence molecules is obtained only in the focus plane where the photon density is high enough. This results are in a far better resolution compared to the fluorescence microscopy.
Another advantage of this technique is the induction of two imaging effects. On the one hand simple autofluorescence (AF) of the sample occurs and on the other hand second harmonic generation (SHG) is induced. SHG just takes place on organized structures (e.g., crystals or collagen). Therefore, in cutaneous probes it is possible to distinguish between AF of epidermal material and the SHG-signal of the collagen in the dermis. This allows collecting information about skin morphology and conditions like skin cancer.
Fluorescence lifetime imaging microscopy
Based on Photon microscopy (either 1- or 2PM) it is possible to measure the lifetime of the excited fluorescence of the molecules, which is the average time between absorption and emission of a photon (FLIM). As the number of photons is linearly proportional to the fluorescence intensity, the fluorescence lifetime can be calculated from the fluorescence intensity decay as 1/e of the maximum fluorescence intensity value. In tissue the fluorescence decay is often multi exponential due to the different molecules. A bi-exponential fit function was used as to be seen in equation 1.1.
Figure 1.6: Principle of the 2-photon and Raman effect: In a) the 2-photon effect is shown. In the 1-photon effect a photon is absorbed by a molecule that pumps the molecule to the excited state. When the molecule drops down to the ground state it loses the energy as fluorescence. In the 2-photon effect the molecule is pumped to the excited state by two almost simultaneously exciting photons with half the energy compared to the 1-photon effect. In b) the Raman effect, the Stokes scattering respectively, is demonstrated. The photon interacts with the molecule, the molecule is pumped to a virtual state and by dropping down to the ground state a photon with a lower energy is emitted. The figure is based on [140, 25].
I0 is the initial intensity of the fluorescence at the time t = 0 and t is the current time. τ1 and τ2
are the fluorescence lifetimes of the fast and slow components. a1 and a2 are the corresponding
amplitudes of the fast and slow components. Looking at the average lifetime of the fluorescence (τm), which is defined in equation 1.2) it is possible to get an information about the molecules and
the surrounding of the molecules and therefore the skin physiology, which is due to the changes in the lifetimes characteristics [121]. Certain particles may also show a fluorescence and therefore a determination of sample morphology and particle can be possible. By following the lifetimes in the z-axes a penetration profile of particles can be created.
I0(t) = I0(a1e − t τ1 + a2e− t τ2) (1.1) τm= (a1τ1+ a2τ2)/(a1+ a2) (1.2) 1.5.2 Raman spectroscopy
The Raman spectroscopy is one the most important methods to investigate materials in physics. It is based on the so called Raman effect which is named after its discoverer the Nobel Prize winner (1930) Sir Chandrasekhara Venkata Raman [117]. It is an inelastic scattering of a monochromatic light beam which is focused into the sample and provides information about the material properties. More in detail a laser is used to examine the sample. The photon interacts with a molecule and
1.6 Biological response to tissue damage (Cytokine release) can either be scattered without an energy transfer (elastic or Rayleigh scattering) or scattered with an energy transfer (inelastic or Raman scattering). The photon energy (E) is related to the Planck constant h 6 and the photon frequency ν (see equation 1.3). There are two ways of a photon
interaction with the molecule in the Raman scattering. This can be seen in b) in figure 1.6). It can interact with the molecule and loses energy (Stokes scattering) which means a higher photon wavelength will be scattered. Another possibility is that the photon enhances its energy (Anti-Stokes scattering). The scattered photons are detected and can be plotted as the intensity over the wavenumber (˜ν= 1/λ) [30]. Each material has its own characteristic spectrum, which makes it possible to identify it. For example, by changing the focus plane of the emitting laser beam it is possible to detect a material penetration in the skin. Overall the Raman spectroscopy is a powerful tool in dermatology for investigating the penetration of Raman-active substances and even metals for example AgNP [12, 162].
E = hν (1.3)
Surface enhanced Raman scattering
Some metallic particles do not show a Raman-shift but there is another interesting effect to visualize those particles by means of Raman spectroscopy. If a molecule is close to Au or Ag the surface-enhanced Raman scattering (SERS) effect can occur. This very rare effect enhances the Raman signal of the molecule drastically based on electromagnetic field enhancement and chemical enhancement near the surface, which results in a well distinguishable spectrum compared to a normal skin background spectrum [54]. Hence, thanks to a "catabolic" enhancer, the penetration of, e.g., silver or gold particles can be clearly investigated [63, 71]. This effect is very sensitive and widely used for detection of trace concentrations, which cannot detected by other less sensitive methods.
1.6 Biological response to tissue damage (Cytokine release)
When exogenous agents as bacteria, viruses or NP come into contact with the skin and therefore with skin cells, the keratinocytes activate cellular responses. One of these responses are the release of cytokines. These are small peptides and can be seen as an important signaling system for the communication between cells. These proteins are building a group of growing and differentiation factors, which are responsible, i.a., for the differentiation of the cells. The cytokines act also, e.g., as inflammatory regulators, induce apoptosis or they recruit immune cells [7]. Important cytokines in this work are IL-1α, IL-1β, IL-6 and IL-8. IL-1α is the most potent pro-inflammatory cytokine in mammals and is build constitutively by keratinocytes [156]. Keratinocytes produce IL-1β in cases of pathogens or bacteria and regulate the balance between cell death and proliferation. They should
6
not be detected in healthy organisms [156]. Damage of tissue, infections and bacteria can induce the release of IL-6 by keratinocytes, which is activating lymphocytes [52]. Another pro-inflammatory cytokine is IL-8. It is also inducing chemotaxis, recruits granulocytes and promotes angiogenese [59].
1.7 Radical formation
One of the most common cause of cytotoxicity are free radicals. These are atoms or molecules, which possess at least one unpaired electron and showing paramagnetic properties (see section 1.9). The formation of free radicals and, above all reactive oxygen species (ROS) is an ongoing physiological process in the human body and in all aerobic cells. During the metabolism, especially in the respiratory chain, ROS such as hydrogen peroxide (H2O2), superoxide radical (O2·-) or
hydroxyl radical (OH-) are frequently produced (see figure 1.7) [95]. ROS also act as a second
messenger such as H2O2as activator for lymphocytes as reviewed by Reth [120]. ROS are involved
in warding off pathogens or used in the photodynamic therapy for killing cancer cells [16]. However, depending on the amount of the ROS, it can be distinguished between helpful, essential ROS and detrimental ROS, which can cause major damages to the body, when they are not regulated. As a counterpart the antioxidant system (AOS) neutralizes the free radicals but in unhealthy or stressed cells sometimes the AOS is not sufficiently to quench them and repair damage. If the system is not balanced oxidative stress occurs. Thus, these highly reactive molecules can cause cellular damage such as membrane degeneration, desoxyribonucleic acid (DNA) damage [153], oxidation of proteins, enzymes and amino acids [137]. The body can provide repair mechanisms such as DNA repair enzymes [84] or degradation of damaged proteins [53]. If the damage cannot be repaired, cells go into apoptosis. Nevertheless nonreversible damages can also lead to uncontrolled cell proliferation, i.e.cancer [143].
1.8 Antioxidant system
Healthy cells keep an equilibrium between oxidative and antioxidative parts. As described in section 1.7 free radicals are not only a side product of the metabolism, they are also essential for many important reactions. Nevertheless, too high amounts of ROS are dangerous for the cells, if they are not quenched in time. Additionally, other reactive species such as reactive nitrogen species or lipid oxygen species can occur and cause cytotoxicity [161, 45]. Therefore the cell uses a system of antioxidants to neutralize these highly reactive radicals.
1.8 Antioxidant system
Antioxidant agents
This part of the defense system does not only neutralize the free radicals into nonhazardous molecules, some of them transform highly reactive ROS into moderately reactive ROS that will be deactivated by other antioxidants. The most important endogenous antioxidants are shortly explained as follows:
Figure 1.7: Scheme of ROS reactions in a cell: The major way of the ROS production is the respiratory chain in the mitochondria from where the superoxide anion (O2·-) is released
through NADPH oxidase (NADPHX) or the release of hydrogen peroxide (H2O2) by
oxidases (X) of the peroxisome. (O2·-) reacts to H2O2via superoxide dismutase (SOD)
reacting to oxygen and water by catalase. Another option is the degradation of H2O2by
glutathione peroxidase (GPX), which uses 2 glutathione (GSH) monomeres as electron donator. The two monomers form a glutathione disulfide (GSSG), which can be recycled by glutathione reductase (GSHR). Figure is based on [93, 95].
• Superoxide dismutases (SOD): This enzyme transformes the superoxide radical to H2O2and
molecular oxygen.
• Catalase: H2O2is decomposed by catalase to water and oxygen.
• Glutathione peroxidase (GPX): Another way to neutralize H2O2 is the GPX which uses
glutathione (GSH) as cofactor.
1.9 Detection of oxidative stress
It comes to oxidative stress when the AOS is overwhelmed. This overproduction of free radicals or the production of radicals at all can be detected by different methods. Most of the systems are based on molecules which interact with free radicals and are transformed in fluorescing dyes. A well-known assay and the gold standard for oxidative stress is the so called dichlorofluorescein-acetate (DCF) assay. The acetate passes the cell membrane and gets de-esterified to its alcohol. This alcohol reacts with free radicals and gets fluorescent [146]. This procedure is established and working well with several types of investigatory systems. But several publications addressed the drawbacks of this assay such as no interaction with H2O2. Another drawback is the possible self-amplification of the
fluorescence due to an interaction of a precursor of DCF with oxygen which results in the formation of H2O2[66]. Additionally and more obvious is that this assay is based on optical measurements
which may lead to interferences with particles or other dyes.
Electron paramagnetic resonance spectroscopy
Another highly sensitive method to detect oxidative stress is the electron paramagnetic resonance (EPR) spectroscopy. EPR is comparable to the magnetic resonance imaging (MRI)7. Unlike the MRI in which the spin of the hydrogen atoms is measured, in the EPR spectroscopy the focus is on the spin of unpaired electrons [149]. Here are paramagnetic molecules, such as free radicals, detected. The main applications can be found in physics and chemistry. Over the years the EPR spectroscopy has progressed and is now a technique used in biological research as well [17]. Based on this concept the measurement of ROS or other free radicals8in cells or tissue is possible. As the free radicals have a short lifetime and cannot directly be detected by EPR spectroscopy, they need to be stabilized by the help of special agents called spin traps or visualized by spin makers [60]. This method is based on the Zeeman-effect (see equation 1.4). Paramagnetic molecules have one or more unpaired electrons. If a magnetic field (B0) is applied the electron energy splits into two
energy states. Either it stays in the parallel state or the antiparallel state as seen in figure 1.8. Energy is absorbed by the unpaired electron when energy is applied in the form of microwaves and fulfills the equation for the Zeeman effect. The energy of an electron is given as the product of h and ν as frequency. If the resonance conditions are fulfilled one get the resonance equation with µB9
as magnetic moment, g10as material-specific gryoscopic constant and B0as the magnetic field.
As the microwave power is measured in the EPR device, this absorption is detected as the first derivative. As seen in figure 1.9 a specific spectrum is obtained. As example the spin marker 2,2,6,6-Tetramethyl-1-piperidinyloxyl (TEMPO) was used. The lines of the spectrum are due to
7
Medical imaging technique to visualize tissue and organs.
8Other radicals are for instance reactive lipid, nitrogen or sulfur species 9
The Bohr magneton expresses the magnetic moment of an electron.
10
1.9 Detection of oxidative stress the interaction of the free electron with the neighboring magnetic nuclei and can be determined by the equation 1.5. This is called the hyperfine coupling with n as the number of nuclei interacting with the electron and l for the nuclear spin [60]. The free electron in TEMPO interacts with the one nucleus of nitrogen and which spin is 1. Therefore the EPR transition is split into three lines.
E = hν = µBgB0 (1.4)
N umber of lines = 2nl + 1 (1.5)
Figure 1.8: Basic concept of the EPR spectroscopy: A magnetic field is applied and the free electron splits into two states (parallel and anti-parallel). Electromagnetic radiation with a micrometer wavelength (microwave) is focused to the sample and if the microwave energy fulfills the resonance equation, the energy is absorbed. This absorption can be measured. Modified after [149].
Figure 1.9: EPR-spectrum of the spin marker TEMPO: Spectrum was obtained using an X-band EPR spectrometer (Magnetech).
1.10 Aim of this work
1.10 Aim of this work
The human skin, as the outer boundary of the body, has developed an effective barrier against external risks such as bacteria, viruses or nano-scaled particles (NP) [13, 89]. The interactions between NP and skin are of interest for biologists, physicians and developers of those particles as nano-scaled materials are associated with a lot of innovative applications but also represent potential risks either [56]. NP have come more and more in the focus of researchers because of their high surface to volume ratio and therefore their high reactivity [125]. In times of rising cases of sepsis due to antibiotic-resistant bacteria, new treatments to reduce this threat are indispensable. Silver NP (AgNP) combine the antibacterial benefits of silver with the advantages of NP. The particles release highly reactive Ag+ions in a continuous and slow manner. The Ag+ions are the reason why Ag and AgNP are used in medicine in a wide range of products since decades [4].
Former investigations on NP-skin-interaction showed so far no clear evidence of a NP (≥40 nm) penetration into the living skin layers [13]. If an uptake was demonstrated, the skin was either injured or changed in its stability (Franz diffusion cell). The uptake into cells and the influence on the cell viability are known from other investigations [48]. AgNP can enter cells and were even found in cell nucleus [10]. AgNP and furthermore the released Ag+ ions can induce a radical formation, which can cause cell damage [26].
In this work, the interaction of NP with human skin was investigated using new measuring techniques and different NP. We intended to understand (i) how and to which extent NP penetrate into human skin, (ii) if and how they exhibit cytotoxic effects and (iii) if and to which extent they cause oxidative stress within the skin cells. It is of interest if the particles in this work (AuNP, AgNP), which differ in material, size and shape, might overcome the skin barrier and interact with skin cells. Even if the skin barrier is not passed, a possible uptake of the particles was investigated as well as the cellular response of AgNP that are widely used in cases of wounds and open lesions and therefore interact with skin cells. Due to this interaction the cell viability and the inflammatory activity can be influenced depending on the type of AgNP, the concentration and the time of incubation. Cytotoxicity of NP is often associated with the formation of free radicals [21, 113]. Therefore the oxidative system should be measured. Hence following major topics were investigated:
1: Penetration of NP into skin and uptake by epidermal cells
Micro-scaled Ag can break down over time and end in AgNP and Ag+ions. That is of high interest as many products on the market use micro-scaled Ag (e.g., particles, crystals, filaments). Many investigations were performed on the penetration of AgNP into the skin. Here methods were used that can mostly ease the penetration of particles (Franz diffusion cell) or remove the particles during preparation (Transmission electron microscopy (TEM)). Hence methods which can avoid
these drawbacks are used. Here, the penetration of NP across intact and disrupted skin barriers should be investigated dependent on their material, size and shape. AgNP can dissolve and lose their shape and size [88]. Determining if the shape is of importance for the penetration of NP, stable and non-dissolving gold NP (rods and spheres) were used additionally. The penetration was investigated on different full skin models (human skin, porcine skin) by means of e.g., confocal Raman microscopy, surface enhanced Raman spectroscopy and fluorescence lifetime imaging microscopy. The next step focuses on the uptake of AgNP (spheres and prisms) into cells (HaCaT cells and human mesenchymal stem cells). Therefore, several techniques were used such as TEM and scanning transmission X-ray microscopy.
2: Influence of AgNP on the cellular viability and on the release of inflammatory mediators
The cytotoxic effects of AgNP are an important question as NP can influence i.a. cell viability [118]. The reaction of HaCaT cells on AgNP was therefore investigated. Here, a cell viability assay was used to see the effect of different concentrations of AgNP on viability and proliferation of cells for different points in time of incubation. It is well known that cytokines are also prominent mediators for stress and damage of the cells [58]. For this purpose different enzyme linked immunosorbent assays (ELISA) were performed to determine possible inflammatory responses due to AgNP incubation.
3: AgNP induction of free radicals and status of the antioxidant system
One major cause of cytotoxicity is the increased formation of free radicals [93]. This highly reactive species can cause cell damage and are responsible for skin aging, wrinkles and in the worst case for cell death [143]. Additionally to the commonly used radical detection assay (DCF assay) EPR spectroscopy was used to measure intracellular radicals. Also the level of glutathione (total, reduced and oxidized form) was determined to acquire knowledge about the antioxidant status of the cells.
1.10 Aim of this work
Figure 1.10: Work plan of this PhD study: The work is divided in three different parts. The uptake of NP, the toxicity and the induction of oxidative stress. Human and porcine skin were used as investigated models as well as human skin cells. Abbreviations are as follows: scanning X-ray microscopy (STXM), confocal Raman microscopy (CRM), fluorescence lifetime imaging microscopy (FLIM), transmission electron microscopy (TEM), cell viability assay (XTT), electron paramagnetic resonance spectroscopy (EPR), dichlorofluorescein-acetate assay (DCF), glutathione assay (GSH), enzyme linked immunosorbent assay (ELISA), gold (Au), silver (Ag).
2 Methods and materials
2.1 Materials
Table 2.1: Devices and technical support
Device Type Manufactor
2-photon microscope DermaInspect Jenlab
Centrifuge Biofuge Heraeus
Centrifuge Multifuge Thermo Scientific
EPR spectrometer Miniscope MS400 Magnetech
Freezer (-80 °C) Fryka
Fridge/Freezer (4 °C, -20 °C) National Lab
Incubator HERAcell Kendro Laboratory Products
Light microsocope IX 50 Olympus
Micropipettes pipetman Gilson
Microplate reader 2300 EnSpire Perkin Elmer
Neubauer counting chamber Fein Optik
Microtom Microm HM 560 Thermo Scientific
Pipettor pipetus®akku Hirschmann
Laborgeräte
Raman microscope Model 3510 Skin River Diagnostics
Composition Analyzer
Safety cabin HERAsafe Kendro Laboratory Products
Scale Analytic balance 770 Kern
Scanning transmission X-ray Bruker
microscope
Secretion suction device P 7010 Medap
Sertological Pipettes 5, 10 and 25 mL Falcon
Timer TFA Dostmann
Transmission electron microscope EM906 Zeiss
Ultrasonic bath Sonorex Super Bandelin
Ultrasonic processor 100H Roth
UV lamp TH-1E Cosmedico Medizintechnik
UVA lamp dermalight®80 uv-a Dr. Hönle
Medizintechnik
Table 2.2: Media, Solvent and chemicals
Product Manufactor
AgNP prisms Free University Berlin
AgNP spheres University Duisburg-Essen
AuNP rods Free University Berlin
AuNP spheres BBI Solutions
DMSO Sigma Aldrich
Ethanol (100%) Herbeta Arzneimittel
Fetal calf serum Biochrom
Glutamine Biochrom
PCA Sigma Aldrich
Penecillin/Streptomycin Biochrom
PBS Biochrom
RPMI-1640 medium Gibco - Life Technologies
TEMPO Sigma Aldrich
Tissue Tek Sakura Finetek Europe
Triton-X Sigma Aldrich
2.1 Materials Table 2.3: Consumables
Product Type Manufactor
Aluminium foil Roth
Cannulas Braun
Capillaries 50 µL Hirschmann
Laborgeräte
Carbon grids Cu 400 mesh Quantifoil Micro Tools
Cell culture flasks T 75 Thermo Scientific
Cell Scraper Costar
Cell viability assay Cell Proliferation Roche Diagnostic
Kit (XTT)
Conical tubes 15 and 50 mL Falcon
Cytokine assay Multi-Analyte ELISArray Quiagen
Gloves VWR
GSH/GSSG assay Glutathione GSH/GSSG Ratio AAT Bioquest
Assay Kit
Haematocrit Sealing Compound Brand
IL-1α ELISA Human R&D Systems
IL-1α/IL-1F1 DuoSet
IL-1β ELISA Human R&D Systems
IL-1β/IL-1F2 DuoSet
IL-6 ELISA Human IL-6 Cytoset Invitrogen
IL-8 ELISA Human IL-8 Cytoset Invitrogen
Lancet needle Alk Abello
Parafilm Bernis
Pipette tips 20, 200 and 1000 µL Roth
Precision Wipes Kimtech
Radical formation assay H2DCFDA (H2-DCF, DCF) Invitrogen
Reaction tubes 1.5 and 2 mL Sarstedt
Scalpel Disposable Feather
Silica grids Silson
Sterilfilter Mesh size 22 µm Roth
Syringe 20 mL, Luer-Lok Braun
Tape Tesa
tGSH assay Amplite Fluoremetric Glutathione AAT Bioquest
Tissue culture plates 96-well SPL Life Science
Tissue culture plate 6-well Falcon
Table 2.4: Properties of the used nanoparticles
NP Coating Batch c / Size by SM/TEM / Zeta-potential /
mg/mL nm mV
AuNP rods PSS 1 3 247 x 22 -37
(lenght x width)
AuNP spheres CTAB 1 3 80 -49
AgNP (O2) PVP 1 0.4496 70 -25 PVP 2 1.326 70 -25 AgNP (Ar) PVP 1 1.266 70 -25 PVP 2 0.9288 70 -25 AgNP prisms PVP 1 0.08 32 -38 (edge to edge)
2.2 Nanoparticles
In this work two different materials were used to investigate the uptake of the particles - gold and silver. The particles also differ in shape and coating. The toxicity experiments were done exclusively with AgNP. Here, NP prepared by different types of synthesis and storage methods were compared. The characteristics of the particles can be found in table 2.4.
2.2.1 Synthesis of silver nanoparticles
The AgNP spheres investigated in this work were kindly provided and characterized by Kateryna Loza and Jörg Diendorf from the Epple group (Institute of Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE), University of Duisburg-Essen, Essen, Germany). The NP were prepared as follows: silver nitrate was reduced with glucose in the presence of polyvinylpyrrolidone (PVP)1according to the method of Wang et al. [147] as described in greater
detail in Kittler et al. [70]. AgNP spheres were prepared under an oxygen (O2) atmosphere and
under argon (Ar). The particles under an Ar atmosphere were aliquoted in vials and overlaid with Ar. The particle characteristics can be found in table 2.4 and were determined as follows: The silver concentrations were measured by means of atomic absorption spectroscopy (AAS; Thermo Electron Corporation M-Series 4110ZL, Perkin-Elmer, Santa Clara, CA, U.S.A.). The size was determined by scanning electron microscopy (SM; Quanta 400 ESEM, FEI, Hillsboro, OR, U.S.A.).
2.2.2 Synthesis of gold nanoparticles
The AgNP rods were kindly prepared and characterized by Daniel Nordmeyer from the Rühl group (Physical and Theoretical Chemistry, Free University Berlin, Berlin, Germany) in accordance with the publication of Nikoobakht et al. [103]. These AuNP rods are coated with cetrimonium bromide
1
2.3 Penetration and uptake of nanoparticles
Figure 2.1: TEM images of AuNP rods: High resolution image of the AuNP rods imaged by Daniel Nordmeyer (Free University Berlin). AuNP rods had a size of approx. 247 x 22 µm.
(CTAB)2and were then wrapped with poly(sodium 4-styrenesulfonate) (PSS)2[109]. The particles were purified and resuspended to remove excess CTAB [50]. The AuNP spheres were purchased from BBI Solutions (Cardiff, U.K.) and no conditioning occurred. The two types of AuNP were characterized by SM (SU 8030 SEM, Hitachi, Tokyo, Japan), the concentrations were adjusted by dynamic light scattering and also the zeta potential was measured (Delsa Nano C, Beckman Coulter, Fullerton, CA, U.S.A.). The characteristics can be found in more detail in table 2.4.
2.3 Penetration and uptake of nanoparticles
The uptake of the NP into the skin and the skin cells was investigated with different methods on different skin models. The performed experiments are summarized in table 2.5.
Table 2.5: Investigations on the uptake of nanoparticles
NP Target Used c of NP / µg/mL Method
AuNP rods Human skin 3000 STXM
AuNP spheres Human skin 3000 STXM
AgNP (O2) Human skin 1326 TEM
HaCaT 25 STXM, TEM
hMSC 10, 25 TEM
AgNP (Ar) Porcine Skin 1266 FLIM, CRM
HaCaT 25 TEM
hMSC 10, 25 STXM, TEM
AgNP prisms HaCaT, hMSC 10, 25 STXM, TEM
2
2.3.1 Skin models
Two skin models were used to investigate the uptake of NP into the skin. One is the porcine skin model and the other one is excised human skin. The preparation of these models was done as follows:
Human skin
Excised human skin was chosen as a well-known and often used model for cutaneous investigations [79, 49]. The skin was a leftover of plastic surgeries such as abdominal reduction or mammalian surgeries. It was cleaned with phosphate buffered saline (PBS) and cut into 2 x 2 cm pieces. The subcutaneous fat was partially removed and the skin was prepared for incubation in a humid chamber (see figure 2.2). A Styrofoam pad was wrapped in aluminum foil and Parafilm. The skin was stretched with cannulas. A marked area of 1 x 1 cm was treated with 20 µL of NP (concentrations are given in 2.4). Afterwards the positive controls were pricked with a lancet needle afterwards to destroy the skin barrier. The skin sample was stored in a humid chamber i.e a plastic box with wet tissues covered with a cap. This chamber was placed in the incubator for 1 h at 37 °C, 5 % CO2
and 100 % humidity. Following that a tape was stuck on the skin and stripped. So the upper layer of the corneocytes was removed; aggregated NP sticking on the surface of the removed tape [81]. After this the marked area was cut out and the remaining fat was removed. Thereafter, the skin was prepared for the next investigative steps:
• Cryo sections:
– Skin is cut into 25 x 25 mm pieces
– Samples are shock frozen in liquid nitrogen – Samples are stored at - 20 °C
• TEM:
– Skin is cut into small pieces (1 mm2)
– Samples are given in bottles (2 mL), which are filled with a fixative agent (2.5 % glu-taraldehyde and 0.1 M Na-cacodylate buffer)
– Samples were stored at 4 °C • STXM
– Skin was cut into small pieces (1 mm2)
– Samples were plunge frozen in liquid ethane and placed in tissue freezing medium – Samples were cut in 3 - 5µm thick slices and transferred on double folding mesh
Cu-TEM grids for freeze-drying or on silicon nitride membrane windows for a wet chamber, respectively
2.3 Penetration and uptake of nanoparticles
Figure 2.2: Humid chamber: Skin samples are fixed on a Styrofoam pad and transferred to the humid chamber. The chamber is filled with wet tissues (a) and covered with a cap (b) to be stored in the incubator.
Porcine skin
Porcine skin and especially porcine ear skin is a widely used skin model [64]. Due to the easy access to this model and the good comparability to human skin is the reason why it is often chosen as model for repetitive investigations such as TEM or FLIM. The pig ears were delivered by a local slaughterhouse and are taken from freshly slaughtered pigs and stored continuously at approx. 7 °C. The ears were cleaned of dirt and the ear conch was removed. After that approx. 4 x 4 cm of the skin was removed with a scalpel. The skin was carefully removed from the conch. This piece of skin was now treated like the human skin as described in chapter 2.3.1, and prepared for the humid chamber. AgNP (Ar) were taken at a concentration of 1.2 mg/mL. 40 µL were applied on 4 cm2of skin. The
control skin was treated with PBS. The samples were stored in the incubator for 24 h in the humid chamber. After this time, the tape stripping procedure was applied once. Now, the porcine skin was suitable for further investigations.
2.3.2 Cultivation of cells
Two cell types were investigated to determine the interaction with NP. HaCaT cells are widely used as a model for keratinocytes and can therefore be taken to perform most of the investigations. Additionally human mesenchymal stem cells (hMSC) were used for STXM (Prepared by Jun.-Prof. Dr. Christina Sengstock, Ruhr-University Bochum, Bochum, Germany).
Cultivation of HaCaT cells
The HaCaT (Human adult low Calcium high Temperature) cells were grown in 75 cm2flasks in 10 mL RPMI-1640 medium (RPMI; Gibco, Life Technologies, Carlsbad, CA, U.S.A.)3with phenol red, 10 % fetal calf serum (FCS), 2 % glutamine, 1 % streptomycin and penicillin. The cells were cultivated in an incubator at 37 °C in 100 % humidity and 5 % CO2. The cells were split every 2 - 3
3
days at a ratio of 1:10 before they reached a confluency of 100 % and started to differentiate. The work was done under sterile conditions in a safety cabin (HERAsafe®, Kendro Laboratory Products). Therefore the RPMI medium was removed at a confluency of approx. 70 - 80 % and the cells were washed with PBS to remove waste products such as dead cells or metabolites. 5 mL of a digesting enzyme solution (Trypsin (0.2 %)4 and ethylenediaminetetraacetic acid (EDTA, 0.02 %)) were added after removing the PBS. After 5 min, cells were detached from the flask as further incubation with trypsin would cause cell damages, hence, the reaction was stopped immediately with the same volume of RPMI medium supplemented with 10 % FCS. The FCS offered an oversupply of substrate, so the protease trypsin did not longer digest cell proteins and adhesion proteins. The cells finally detached by gently knocking at the flask. Additionally, a cell scraper could be used. Observing the HaCaT cells under the microscope one could realize that the cells were roundly shaped and smaller than adherent cells. Afterwards, the cells were centrifuged (360 g, 10 min) and resuspended in 10 mL RPMI medium so that the cells could be counted using a Neubauer chamber. The cells were seeded in new flasks (10 mL cell suspension with 1·105cells/mL) or used for further investigations as a final step.
2.3.3 2PM-induced Fluorescence lifetime imaging microscopy
One technique used to investigate the penetration of AgNP into the prepared porcine skin is the 2PM with FLIM. The 2PM (DermaInspect, Jenlab, Jena, Germany) is equipped with a tunable femtosecond titanium sapphire laser (710 - 920 nm) running at 760 nm. The laser is focused on the sample within a volume of a femtoliter by means of an oil-immersed objective (magnification 40 x). The scanning is ranged in X-Y-Z direction at 350 x 350 x 400 µm. The 2PM images provide a resolution of 512 x 512 pixels, the FLIM images 128 x 128 pixels by a lateral resolution in skin of 0.4 -0.6 µm and an axial resolution of 1.2 - 2.0 µm. The FLIM analysis was done with a DermaInspect incorporated software (SPCImage 4.2).
The measurement was performed by fixing the porcine skin sample on the cover glass. The skin surface was searched and set as zero. A stack of 2 µm steps was performed down to 18 µm within an acquisition time of 9.6 s for each image. The FLIM curve is always approximated biexponentially i.e.by the quick and slow exponents.
2.3.4 Confocal Raman microscopy
The porcine skin samples were prepared as described in section 2.3.3 and transferred to the confocal Raman microscope (CRM; Model 3510 Skin Composition Analyzer, River Diagnostics, Rotterdam, The Netherlands). For the measurements a near-infrared laser (785 nm, 26 mW) was used to investigate the samples in the fingerprint region (400 - 2000 cm-1). The chosen wavelength is frequently used for skin investigations due to the skin’s reduced absorption and scattering and hence
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2.3 Penetration and uptake of nanoparticles its proper penetration ability [22, 35]. The spectra were recorded from the surface to a depth of 40 µm at steps of 2 µm. The acquisition time for each spectrum was 5 s. 10 different points were measured for each porcine ear skin sample. All in all, 3 skins from animals were used to investigate the penetration of the AgNP into the skin. The penetration profile was determined by the non-restricted multiple least square fit method5using the Skin Tools 2.0 software (River Diagnostics).
2.3.5 Surface enhanced Raman scattering
The measurements were done using the same procedure as described for the CRM (see section 2.3.4). Three different porcine ears were used and 10 different points were measured at each skin sample. Excitation wavelength of 785 nm and a laser power of 26 mW were used. Spectra from the skin surface were obtained down to 40 µm in 2 µm steps. A SERS signal appears only if skin stays in contact with AgNP. The presence of the SERS signal served as criteria for the presence of AgNP in the skin layer.
2.3.6 Cryo sections
For some microscopic techniques used in this work it was important to get thin sections of the samples. Therefore histological sections were prepared. For this classical method the tissue is dehydrated and fixed with paraffin. In this work another method was used - the cryo section procedure. Frozen samples were used, which were prepared as described in section 2.3.1. A cryostat Microm HM 560 (Thermo Fisher Scientific, Waltham, MA, U.S.A.) was used to cut the sample into sections. This microtome did hold a specific temperature and was usually operated at - 20 °C. First, the sample was embedded in a gel (Tissue Tek, Sakura Finetek Europe, Alphen aan den Rjin, The Netherlands), which freezes rapidly on the - 50 °C cold freezing plate and the density was almost the same as the frozen skin tissue. The sample was mounted and cut with a scalpel to the right shape. With a size of 30 µm first sections were cut until the sample and the tissue were visible. From here on out the size was changed in 3 - 6 µm dependent on the sample. The section was picked up on a microscope slide and checked with light microscopy (IX 50, Olympus, Tokyo, Japan). Other steps such as staining can now be done if needed. The transfer of a section could also be done onto special grids for e.g., STXM or TEM . Otherwise the cryo sections could be stored in the freezer at - 20 °C for further investigations.
2.3.7 Transmission electron microscopy
Preparation of skin for TEM
An established method was used to investigate intracellular uptake and the skin morphology - the transmission electron microscopy (TEM). The final preparation of the samples was kindly done by
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