The phospholipid 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC) and cholesterol were purchased from Avanti Polar Lipids, Inc. (Alabaster, Al, USA). Sucrose and glucose were purchased from Sigma–Aldrich (Steinheim, Germany).
Titanium (IV) oxide nanopowder (TiO2), supplied as a powder of anatase crystalline structure, with guaranteed 99,7% purity, and zinc oxide nanopowder (ZnO) were purchased from Sigma Aldrich (Steinheim, Germany).
Preparation of engineered particle suspensions
For experiments with washed erythrocytes or platelet-rich plasma, stock suspensions of EPs were prepared in phosphate buffer saline with trisodium citrate (PBS –citrate: 137 mM NaCl, 2,7mM KCl, 10 mM Na2HPO4x2H2O, 2 mM KH2PO4, 10,9 mM Na3C6H5O7) with a concentration of 5 mg/ml. For experiments with GUVs, stock suspensions of TiO2 and ZnO EPs were prepared in 0,3 M glucose with a concentration of 1 mg/ml.
Characterization and imaging of engineered particle suspensions
Characterization of EPs (primary characteristics of EPs) and their suspensions (secondary characteristics of EPs) were assessed by scanning and transmission electron microscopy (SEM and TEM, respectively), dynamic light scattering (DLS) and zeta potential (ζ) measurements. For TEM, water suspension of EPs was applied on a copper-grid-supported, perforated, transparent carbon foil at room temperature. TEM imaging of EPs was performed using a JEOL 2100 transmission electron microscope (Tokyo, Japan) operated at 200 kV. SEM imaging of EPs in PBS-citrate was performed using a LEO Gemini 1530 (LEO, Oberkochen, Germany) scanning electron microscope.
Dispersed TiO2 and ZnO EPs dissolved in 0,3 M glucose (10 μg/ml) were inspected by DLS using a 3D DLS-SLS spectrometer (LS Instruments, Fribourg, Switzerland). Due to very fast sedimentation of both TiO2 and ZnO EPs, DLS measurements in PBS could not be performed. The zeta potential (ζ) of TiO2 and ZnO EPs suspensions in both media, PBS-citrate and in 0,3 M glucose, were measured at the original concentrations and the pH values of the suspensions using a ZetaPals instrument (Brookhaven Instruments Corporation, Holtsville, NY, USA).
Blood was collected from the authors and from 3 healthy pet dogs owned by members of the staff of Prva K Klinika za male živali d.o.o. Ljubljana. 2,7 ml tubes containing 270 μl trisodium citrate at a concentration 0,109 mol/l were used. All the dogs weighed over 15 kg so the volume of the blood sample (2,7 ml) was small compared to the whole volume of the animal. Blood was collected by vein puncture into evacuated tubes (BD Vacutainers, Becton Dickinson, CA) using a 21-gauge needle (length 70 mm, inner radius 0,4 mm) (Microlance, Becton Dickinson, NJ, USA). Sampling was performed according to the Declaration of Helsinki. Informed consent was obtained from all human participants in this study and written consent to take blood from the dogs was obtained from their owners. The study as regarding both human and animal samples was approved by the Slovenian National Medical Ethics Committee, No 117/02/10. No adverse effects on human and animal donors’ health due to sampling were observed.
Preparation of blood cells for microscopy
Blood was processed within 1 hour of sampling. Blood was centrifuged in a Centric 400R centrifuge (Domel d.o.o., Železniki, Slovenia) at 150 g and 37°C for 10 minutes (human) and at 50 g and 37°C for 15 minutes (dog) to separate erythrocytes from platelet-rich plasma. Erythrocytes were repeatedly washed with PBS citrate by centrifugation at 1550 g and 37°C for 10 minutes. Washed erythrocytes or platelet-rich plasma were aliquotted into equal parts (50 μl in case of erythrocytes and 200 μl in case of platelet-rich plasma). TiO2 or ZnO EPs suspended in PBS-citrate (or PBS-citrate alone for the control) was added to aliquot in a v/v ratio of 2:1 in the case of erythrocytes and 3:1 in the case of platelet-rich plasma, 3 and 2 units corresponding to erythrocytes and platelet-rich plasma, respectively. After incubation the samples were fixed in 0,1% glutaraldehyde, incubated for another hour at room temperature and centrifuged at 1550 g and 37°C for 10 minutes. The supernatant was exchanged with PBS-citrate, samples were vortexed, centrifuged at 1550 g and 37°C for 10 minutes and fixed in 2% glutaraldehyde for an hour.
Phase contrast microscopy of erythrocytes
5 μl of erythrocyte suspension was placed in an observation chamber composed of two cover glasses which were glued together with nail polish. Samples were observed under an Olympus GWB BH-2 (Olympus Corporation, Tokyo, Japan) microscope with phase contrast optics at an objective magnification of 400×. Images were taken with a Canon EOS 450D digital camera (Canon Inc., Tokyo, Japan).
Scanning electron microscopy of blood cells
Fixed samples were washed by exchanging supernatant with citrated PBS and incubated for 20 minutes at room temperature. This procedure was repeated 4 times while the last incubation was performed overnight at 8°C. Samples were then post-fixed for 60 min at 22°C in 1% OsO4 dissolved in 0,9% NaCl, dehydrated in a graded series of acetone/water (50%-100%, v/v), critical-point dried, gold-sputtered, and examined using a LEO Gemini 1530 (LEO, Oberkochen, Germany) scanning electron microscope.
Preparation of giant unilamelar phospholipid vesicles
The electroformation of GUVs was performed at room temperature. 40 μl of the lipid mixture of POPC (80%, v/v) and cholesterol (20%, v/v), both dissolved in 2:1 chloroform/methanol mixture, was spread over two platinum electrodes and the solvent was allowed to evaporate in low vacuum for two hours. The electrodes were then placed in the electroformation chamber (a 2 ml Eppendorf cup), filled with 2 ml of 0,3 M sucrose solution. An alternating electric field was applied as described in . After electroformation, 600 μl of 0,3M sucrose solution containing GUVs was added to 1 ml of 0,3M glucose solution.
Experiments with giant unilamelar phospholipid vesicles and engineered particles
Experiments with the two types of EPs, TiO2 and ZnO, were performed separately, but following the same procedure. After electroformation, the suspension of GUVs in sucrose/glucose solution was mixed by turning the Eppendorf cup upside down (five times). The suspension was diluted (300 μl of 0,3M glucose solution was added to 100 μl of original vesicle solution) in order to obtain the desired concentration of GUVs, which was found convenient according to preliminary experiments. Subsequently, the diluted suspension was aliquotted into 4 vials, (81 μl in each vial). Duplicates of test suspension of EPs (TiO2 or ZnO, both dissolved in 0,3M glucose solution with a concentration of 100 μg/ml) and duplicates of control suspension (0,3M glucose solution) were added to GUV suspensions in a volume ratio of 9:1 (GUVs: test suspension) to reach the final concentration of EPs of 10 μg/ml. Vials were turned upside down five times to mix the test suspension with the GUV suspension. The samples (70 μl each) were separately transferred into four CoverWellTM Perfusion chambers PC4L-0,5 (Grace Bio-Labs Sigma-Aldrich, Steinheim, Germany) to create duplicates for the test sample and duplicates for the control sample.
The observation chambers with samples were mounted to the inverted phase contrast light microscope (Nikon Eclipse TE2000-S, Tokyo, Japan). After pre-defined durations of incubation (20 and 50 minutes), GUV populations in each chamber were recorded by a Sony CCD video camera module, model: XC -77 CE (Minato, Japan). These recordings consisted of two video sequences each approximately 2 minutes long. During recording, the object glass was moved to capture images of thousands of GUVs. Using image processing algorithms, the video sequences were transformed into large mosaics  where each mosaic contained all the vesicles acquired within the sample. GUVs in mosaics were segmented using a computer-aided approach .
Here we introduced improvements to the analysis of GUV populations. The mosaics were separated into images (micrographs) with the size of a single field of view with the microscope at a given magnification. Using the GUVs’ locations inside the mosaics, all vesicles were associated with the appropriate micrograph, allowing us to extract information on vesicle density throughout the micrographs composing the mosaic.
The numbers of GUVs in a micrograph were averaged over all micrographs within the test and control samples. Methods of descriptive statistics were used to compare the test samples and the respective control samples. We used a two-sided pooled t-test with equal variance. A probability of the t-test below 0,05 was considered statistically significant. Power analysis was performed in order to validate the size of the samples. A power larger than 0,9 at α = 0.05 indicated a sample of proper size. The software used for statistical analysis was from R Development Core Team: R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. 2008 ISBN 3-900051-07-0, URL http://www.R-project.org.