Preparation of silver (Ag) NPs
50 nm bare and PVP-coated AgNPs were synthesized according to Bastús and co-workers [43]. 1 ml of 0.5 M sodium citrate and 1 ml of 25 mM tannic acid were mixed with 97 mL H2O in a three-neck round-bottomed flask. The mixture was heated to boiling with vigorous stirring followed by fast injection of 1 ml 50 mM AgNO3. NP growth was achieved by consecutive addition of 50 mM AgNO3 (1 ml per addition). After each injection, the solution was kept under reflux to complete the reaction for 30 mins. 50 nm spherical AgNPs were obtained at the 10th injection. The as-prepared NPs were centrifuged at 8000 x g for 15 min prior to conjugation with PVP.
Conjugation of silver nanoparticles with polyvinylpyrrolidone
Synthesized AgNPs (∼50 nm, 7.5 × 1011 NPs/mL) were redispersed in a fresh solution of 5 mM polyvinylpyrrolidone (PVP, MW = 55 kDa) and vigorously stirred for 72 h. Then, the AgNPs were washed again to eliminate excess PVP.
Synthesis of Ag nanocubes
Ag nanocubes were synthesized as in [44]. Briefly, ethylene glycol (5 ml; EG) was heated with magnetic stirring in a 100 ml round-bottomed flask in an at 150°C. Sodium hydrosulfide (NaSH; 0.06 ml; 3 mM in EG) was quickly injected into the solution after its temperature reached 150°C. After 2 min incubation, 0.5 ml aliquots of 3 mM HCl in EG and then 1.25 ml PVP (20 mg/ml in EG, MW 360 kDa) were injected into the reaction solution. After another 2 min incubation, silver trifluoroacetate (CF3COOAg; 0.4 ml, 282 mM in EG) was added into the mixture. During the entire process, the flask was capped with a glass stopper except when adding reagents. After addition of CF3COOAg, the transparent solution became a whitish color and then slightly yellow after 1 min, indicating the formation of Ag seeds and then nanocubes.
Synthesis of PVP-coated Ag nanotriangles
PVP-coated Ag nanotriangles were synthesized as in [45]. A 24.04 mL aqueous solution containing AgNO3 (0.05 M, 50 µL), trisodium citrate (75 mM, 0.5 mL), PVP (40K, 17.5 mM, 0.1 mL), and hydrogen peroxide (H2O2; 30 wt%, 60 µL) was vigorously stirred at room temperature in air. Sodium borohydride (NaBH4, 100 mM, 250 µL) was rapidly injected into this mixture to initiate the reduction. The solution gradually turned from light yellow to dark blue within 60 mins.
Synthesis of Ag nanorods
0.5 ml of FeCl3 solution (0.6 mM, in EG) was added to 6 ml EG in a round-bottom flask and was heated to 150 ± 4°C. Then, 6 ml EG solution containing 0.052 M AgNO3 and 0.067 M PVP (average molecular weight 360 kDa) was added. The reaction mixture was kept at 150 ± 2°C with stirring at 250 rpm until AgNO3 was completely reduced (about 70–90 minutes).
To examine the yield and morphology of Ag nanorods, 1 ml of the resulting suspension was diluted with 8 ml acetone and 8 ml ethanol and centrifuged at 2000 rpm for 10 min twice. At every stage, the supernatant solution was measured with a UV spectrometer to confirm the relative amount of AgNPs. All the synthesized AgNPs were washed several times with water and then stored at 2–8°C and protected from light.
Transmission electron microscopy (TEM)
TEM images were obtained with a JEOL JEM 1010 (JEOL Ltd., Tokyo, Japan) and Phillips CM20 (Philips, Amsterdam, Netherlands) at 200 keV using carbon grids (S162, Plano GmbH, Wetzlar, Germany). Carbon grids were dried at room temperature (RT), and the areas of the grid were observed at different magnifications. TEM pictures were computer analyzed in situ and the size distribution and average size of particles were determined.
Dynamic light scattering (DLS) and Z-potential measurement
NPs suspended in water, phosphate buffered saline (PBS), 10% fetal calf serum in PBS, and culture medium were characterized by dynamic light scattering (DLS) and by zeta-potential determination (Malvern Zetasizer Nano ZS90; Malvern, UK). Particles were sonicated for approximately 20 seconds before being dispersed in the appropriate dispersants. All DLS measurements were performed with a Malvern Zetasizer Nano ZS90 (Malvern) operating at a light source wavelength of 532 nm and a fixed scattering angle of 173° on 1 ml aliquots of the NP suspensions. Zeta-potential and DLS assays were performed at 25°C and 37°C and are presented as averages and standard deviations of the data obtained from 3 to 5 assays in each solution.
UV–visible spectrophotometry of AgNPs
UV–visible spectra of 1 ml aliquots of the NP suspensions were assayed with a Shimadzu UV-2400 spectrophotometer in the 300–800 nm wavelength range. This technique provides characteristic absorbance maxima for metallic NPs (due to their surface plasmon resonance), which changes with the size, morphology, and surface alterations of the NPs. UV-vis extinction spectra were taken at room temperature using a 1 cm optical path quartz cuvette by diluting 0.1 mL of sample solutions into 1 mL.
Nanoparticle tracking analyses (NTA)
Nanoparticle tracking analyses (NTA) were performed using a Nanosight LM10 (NanoSight Ltd., Salisbury, UK) equipped with a red laser (630 nm) and CCD camera. The samples were dispersed in milli-Q water, and the experiments were performed at 22°C. The Brownian motion of the particles was analyzed on 60-second records by the NTA software.
Differential centrifugal sedimentation (DCS)
DCS experiments were performed with a disc centrifuge (Model DC 24000; CPS Instruments Europe, Oosterhout, The Netherlands). A gradient of 2–8% sucrose equilibrated with spinning at 22,000 rpm for 30 minutes was established and calibrated by running standard polystyrene beads. After establishment of the gradient, 100 µl aliquots of particles dispersed in water were injected. Samples were spun for approximately 2 hours for PS NPs and 5–10 minutes for spherical AgNPs. The position of particles in the gradient was analyzed with CPS software. The tallest peak (the most frequent size value) was regarded as the ‘base’ peak (100%), and all other particle size peaks were normalized against this base peak (relative size distribution).
Human blood proteins on spherical Ag PVP NPs
In situ protein coronas on spherical Ag PVP NPs were prepared by incubating 0.1 mg/ml NPs in 10%, 80%, and 100% human plasma solution (total protein content 34–47 mg/ml) at room temperature for 1 hour.
The human plasma was obtained from the Centre for BioNano Interactions (CBNI), School of Chemistry and Chemical Biology, University College Dublin, Dublin, Ireland. The blood donation procedure was approved by the Human Research Ethics committee at University College Dublin. The blood plasma was prepared following HUPO BBB SOP guidelines [46]. In brief, after blood collection, the blood was mixed with 2 mM EDTA and centrifuged for ten minutes at 1300 x g at 4°C. Plasma from each donor was collected into 50 ml Falcon tubes and then centrifuged at 2400 x g for 15 minutes at 4°C. The supernatant was collected, aliquoted into 1 ml cryovials, and stored at -80°C until use. Following this procedure, the plasma protein concentration was estimated to be 80 g/l. Before the experiments, the plasma sample was thawed at RT and centrifuged for 3 min at 16,200 RCF. After incubation with human plasma, the NP samples were directly injected into the DCS instrument without spinning down and washing.
NE-4C neuroectodermal stem cell culture
NE-4C neuroectodermal stem cells (ATTC CRL-2925 [47]) were cloned from primary brain cell cultures prepared from the fore- and midbrain vesicles of 9-day-old transgenic mouse embryos lacking functional p53 tumor suppressor protein. NE-4C neuroectodermal stem cells were maintained in poly-L-lysine-coated culture dishes in minimum essential medium (MEM; Sigma Aldrich, St. Louis, MO) supplemented with 4 mM glutamine and 10% fetal calf serum (FCS; Sigma Aldrich) (MEM-FCS).
Cell exposure to nanoparticles
For viability assays, the cells were grown in 96-well plates (104 cells/well) and were exposed to different doses of NPs (from µg/ml;) in serum-free MEM-F12-ITS medium for 24 hours. For uptake experiments, the cells were grown in 24-well plates (105 cells/well) and were exposed to 50 µg NPs (1010 NPs/ ml) in MEM-F12-ITS medium for 1 h. During exposure to NPs, the cells were kept at 37ºC in a 5% CO2 and 95% air atmosphere incubator. The NP dispersions were prepared immediately before use and vortexed before distribution in the culture wells.
Cell viability assays (MTT reduction)
For assessing MTT reduction, an index of cellular activity, we used the redox reaction of the same compound 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). MTT can be reduced to a purple formazan [48], and formazan production can be determined by photometrically measuring the absorption of 550–570 nm wavelength light. The metabolic activity of cells was measured by the MTT reduction assay on living cells [48].
Cells grown on 96-well plates (104 cells/well) were exposed to NP suspensions (from 7.8 to 250 µg/ml) in 100 µl of MEM-F12-ITS. The cells were incubated for 24 h at 37°C in 95% air and 5% CO2 atmosphere. The reaction was stopped by adding 100 µl stop solution containing 50% dimethylformamide and 20% sodium dodecyl sulfate in distilled water (DMF-SDS, pH 4.7). After dissolving the cell material and the formazan product in the stop solution, formazan quantity was determined by measuring light absorption at dual [550–570 nm (measuring) and 630–650 nm (reference)] wavelengths using a Bio-Rad 450 (BioRad Hungary Ltd., Budapest, Hungary) or Dynatech MR5000 (Dynatech Industries Inc., McLean, VA). To obtain comparable data on different cells and culture plates, optical density data measured in each well were related to values obtained on control (non-exposed) cells on the same plate (100%). The data were presented as relative percentages of the control. Averages and standard deviations were calculated from 8–12 identically treated cultures. Significance was calculated with the Student t-test. Differences were regarded statistically significant if p-values were < 0.05 and biologically significant if dose-dependent responses were detected.
TEM analysis of the cellular uptake of AgNPs of different shape
Neural stem cells were grown on poly-L-lysine-coated glass coverslips in 24 well plates (105 cells/well). The cells were incubated with 500 µl suspension of 50 µg/ml (2 x 1011 NPs/ml) NPs dispersed in MEM-F12-ITS for 1 h at 37°C in a CO2 incubator. Control cells were incubated without NPs. The cells were washed three times with PBS (pH 7.4) to remove free-floating NPs and fixed for 20 min with freshly prepared glutaraldehyde 1% and 4% PFA solution before being post fixed in 2% osmium tetroxide (OsO4) in 0.1 M PBS pH 7.4 at 4°C for 2 hours. After washing, the preparations were dehydrated in increasing (30%, 60%, 96% v/v) concentrations of ethanol and embedded in LX-112 resin (Ladd, Burlington, VT). Sections (60–80 nm) were cut with an ultracut (UCT, Leica EM UC7, Wetzlar, Germany) and then contrasted with 1% uranyl-acetate in 50% ethanol and examined by TEM as above.