2 Methods and materials
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
2Stabilizer in nanoparticle syntheses.
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 chamberi.ea 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 (Humanadult lowCalcium highTemperature) 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
3Named after the Roswell Park Memorial Institute
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
4This protease digests the adhesion proteins and intercellular connections.
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 fore.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
5Mathematical approach in regression analyzes for the approximated solution of overestimated systems.
Petra Schrade from the group of Prof. Bachmann (Department of Electron Microscopy, Charité -Universitätsmedizin Berlin, Berlin, Germany). The prepared and fixed skin samples (see section 2.3.1) were refixed in 1 % osmium tetroxide. Afterwards the samples were dehydrated in a washing series at increasing ethanol percentages (50, 70, 80, 95 and 100 %). Now, the samples were immersed for 1 h in a propylene and Epon resin mixture (SERVA Electrophoresis, Heidelberg, Germany) at ratios of 2:1, 1:1 and 1:2. Finally, the samples were incubated overnight in pure resin. After this, the samples were placed in capsules with fresh resin at 60 °C for two days. This resin block was sectioned and stained with Richardson stain6. After identifying the area of interest the useless parts were removed from the resin block. Ultrathin sections (70 nm) were prepared and placed in carbon grids (Cu 400 mesh, Quantifoil, Großlöbichau, Germany). Then the sections were stained with Reynolds lead citrate, which contains lead nitrate (Merck, Darmstadt, Germany) and sodium citrate (Merck). These sections were observed with a Zeiss EM906 microscope (Zeiss, Oberkochen, Germany).
Preparation of cells for TEM
The HaCaT cells (2.5·105cells/mL) were seeded on a 6-well plate and grown for one day. After that the cells were incubated with AgNP in RPMI for another 24 h in the incubator. Then the supernatant was removed, the cells were washed with PBS and afterwards the cells were tripsinized for 5 min.
The trypsin was blocked with RPMI and the cells were centrifuged. The supernatant was removed and the cell pellet was washed with PBS and centrifuged again. The PBS was removed and the cells were resuspended in Fixant. The fixed cells were prepared for the TEM investigations by the group of Prof. Bachmann. Thereafter the cells were mounted in agarose gel (Sigma Aldrich) and prepared for human skin as described.
2.3.8 Scanning transmission X-ray microscopy Preparation of skin for STXM
The skin biopsies were placed in a tissue freezing gel and were cut with a microtome (Thermo Fisher Scientific) at -20 °C in slices of 3 - 5 µm thickness as mentioned in section 2.3.6. The frozen samples were transferred onto silicon nitride membrane windows (Si3N4; Silson, Bliswoth, U.K.).
The slices were heated to room temperature and fixed with 2 % paraformaldehyde (PFA). The PFA was removed and a droplet of distilled water was added. Then, the grid was covered with another grid and sealed with nail polish to form a wet chamber. For the freeze-drying procedure the slices were transferred on a double folding mesh Cu-TEM grids (Plano, Wetzlar, Germany). The samples were freeze-dried cautiously by warming the temperature from -130 °C to room temperature at p = 10-3Pa [50].
6Mostly a mixture of methylene blue and Azure II
2.3 Penetration and uptake of nanoparticles
Figure 2.3:Scheme of the STXM:The scheme shows the endstation of the STXM beamline. The X-ray beam is focused on the sample by a plate zone where the photons are scattered or absorbed by the sample. The non-absorbed photons are crossing the sample and hitting a phosphor screen, which is coupled to a photomultiplier tube (PMT).
The investigation of the prepared samples was done at the PolLux STXM at the SLS (Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland). The storage ring operates at 2.4 GeV. It provides a constant beam current of 400±1.5 mA, which is deflected to the endstation (see figure 2.3). The STXM runs with a linear polarized X-ray beam in the photon energy range between 250 and 1600 eV. The microscope provides a flux of 107photons/s at 300 eV, which is measured using a photomultiplier tube (PMT). The samples are mounted on a piezoelectric stage and scanned horizontally and vertically, perpendicular to the X-ray beam to achieve two-dimensional images.
The beam is focused via Fresnel zone plates to get a spatial resolution of about 40 nm. A phosphor screen, which is coupled to a PMT collects the transmitted signal. Image processing is performed by computer-controlled electronics and the software aXis2000.0 The system is running under a vacuum to minimize X-ray absorption by air. The vacuum also prevents side effects such as sample contamination. Recordings were performed at photon energies below C 1s edge at 270 eV and the O 1s edge at 550 eV.
Preparation of cells for STXM
HaCaT cells and hMSC were prepared to be investigated via STXM. They (2.5·105HaCaT cells /mL or 1.5·104hMSC /mL) were grown on Si3N4-membranes and incubated with AgNP-spheres or rods (25 µg/mL) [130]. After that the grown cells were fixed with PFA. Then one droplet of aqua dest. was poured on the grid, a second grid was used to cover the sample. The edges of the grids were glued together with nail polish to seal it obtaining a kind of wet chamber. This chamber was fixed and investigated on the sample holder at the SLS.