Characterization of SiNPs
Silica (SiO2) nanoparticles (SiNPs) suspensions were purchased from nanoComposix Company (San Diego, CA, USA). The surfaces of SiNPs were coated with silanol by the manufacturer to increase particle hydrophilicity. The tested SiNPs were with two diameters: 20 nm (SiNP-20) and 100 nm (SiNP-100). The size and morphology of SiNPs were assessed by TEM. The hydrodynamic size distribution and zeta potential of SiNPs in double-distilled water or serum containing medium were measured using Zetasizer (Nano ZS90; Malvern, UK). The SiNPs were sterilized and then dispersed with a sonicator for 5 min prior to use. Potential contamination of endotoxin in the SiNPs solution was also examined using the Limulus amebocyte lysate (LAL) gel clot assay kit (Houshiji Company, Xiamen, China), and endotoxin was proved negative in the SiNPs samples.
Primary human umbilical vein endothelial cells (HUVECs), endothelial cell culture medium (ECM), and endothelial cell growth supplements were all purchased from Science Cell Research Laboratories (San Diego, CA, USA). Cells were grown in endothelial cell culture medium (ECM) containing 5% fetal bovine serum (FBS) and penicillin-streptomycin endothelial cell growth supplements at 37 °C in a humidified atmosphere containing 5% CO2. Cultured HUVECs at 80% confluency were used for further studies.
Transmission electron microscopy (TEM)
Uptake of SiNPs by HUVECs and intracellular localization of SiNPs after exposure was examined using TEM. Cells were exposed to 50 μg/mL of either SiNP-20 or SiNP-100 and were cultured in ECM at 37 °C in 5% CO2 for 24 h. Cells were then washed three times with PBS, detached with 2.5% trpsin, then fixed with 2% glutaraldehyde for 2 h. After dehydration with alcohol, cell samples were embedded in epoxy resin. Ultrathin sections were cut using an ultramicrotome and TEM images of HUVECs were taken under a transmission electron microscope (JEM-100CX; JEOL Ltd., Japan).
Cell viability was assessed using cell count kit (CCK-8, Dojindo Molecular Technologies, Inc., Kumamoto, Japan). HUVECs were seeded into each well of 96-well culture plates at a density of 5 × 103 cells/well. After exposure to SiNPs of either size at concentrations of 0–200 μg/mL for 24 h (n = 5), the medium was removed from each well and cells were washed with PBS and incubated for an additional 2 h with fresh medium containing 10 μl of CCK-8 reagent. Then the absorbance of medium was measured at 450 nm using a microplate reader (Synergy-HT, BioTek, Winooski, VT, USA). Data were normalized to the absorbance of the untreated control cells. The viability of control cells was set to 100%.
Cell membrane integrity was assessed by quantifying the lactate dehydrogenase (LDH) released from HUVECs to the medium following pretreatment with SiNPs (0–200 μg/mL) for 24 h using a LDH kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Cell density and experimental grouping were similar with cell viability assay. Each experiment was repeated in 5 parallel wells. Control cells were treated with the culture medium. After incubation for 24 h, the optical density values were measured with a microplate reader.
Fluorescence microscopy to measure intracellular ROS generation
The level of intracellular reactive oxygen species (ROS) was determined in HUVECs exposed to SiNP-20 and SiNP-100 at concentrations of 25, 50 and 100 μg/mL for 2 h (n = 3) using a cell-permeable fluorogenic probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). DCFH-DA diffuses into cells and reacts with ROS to form the highly fluorescent product 2,7-dichlorodihydrofluorescein (DCF). Cells were seeded into a 24-well plate at a density of 1.0 × 104 cells/well and pre-treated with DCFH-DA stock solution for 30 min before exposure to SiNPs. After removing the DCFH-DA solution from all wells, cells were exposed to SiNPs in fresh medium for 2 h. Cells not treated with SiNPs were served as negative control, while cells treated with 7.5 mg/mL H2O2 were positive control. The fluorescence of oxidized DCF was imaged with a fluorescence microscope (Olympus, Tokyo, Japan).
Flow cytometry to measure intracellular ROS generation
Intracellular accumulation of ROS in HUVECs after exposure to SiNPs was detected using flow cytometry (FC500; Beckman Coulter, Hialeah, FL, USA). Briefly, HUVECs were seeded into 6-well plates at a density of 2 × 105 cells/dish and were treated with SiNP-20 or SiNP-100 at 100 μg/mL or without SiNPs treatment (control). After incubation with SiNP-20 or SiNP-100 for 1 h, 2 h, 4 h, 6 h, 12 h and 24 h, cells were harvested and dyed with fresh ECM containing DCFH-DA for 20 min at 37 °C, and were then washed thrice with PBS. The production of ROS was determined using flow cytometry with an emission wavelength of 525 nm (FL1) for DCF. Data were calculated using CellQuestR software (Becton Dickinson, Mountain View, CA, USA) and expressed as mean channel fluorescence for each sample (1 × 104 cells auto-selected from 2 × 106 cells by flow cytometer).
HUVECs migration was evaluated using a transwell system (Corning Costar Corp., Cambridge, MA, USA) which comprised 8 μm polycarbonate filter inserts in 24-well plates. Briefly, cells were trypsin-harvested in ECM with 5% FBS. Next, 600 μL of medium containing 5% FBS without or with SiNP-20 and SiNP-100 at concentrations of 25, 50 and 100 μg/mL was added to the lower chambers, while HUVECs (5 × 104 cells) were plated in the upper chambers. After 12 h incubation, cells migrated to the bottom side of the transwell membrane were fixed with 4% paraformaldehyde at 37 °C for 15 min and stained with 1% crystal violet at 37 °C for 20 min. The non-migrating cells in the upper chamber were removed with blunt-end swabs. The membranes were washed three times with PBS and photographed under a fluorescence microscope (Olympus; Tokyo, Japan). The amount of cell migration was counted under 5 fields. Each treatment was repeated for 3 independent chambers.
Capillary-like tube formation assay
HUVECs tended to form capillary-like structures (tubes) on matrigel. Tube formation was observed and evaluated with confocal microscopy. HUVECs were treated with SiNP-20 and SiNP-100 at concentrations of 25, 50 and 100 μg/mL or without SiNPs treatment (control). Each treatment was repeated for 3 independent dishes. At least 30 min before the experiment, a special dish for confocal study was coated with matrigel (BD Biosciences, Bedford, MA, USA). Next, trypsin-harvested HUVECs were seeded onto the plated matrigel (2 × 105 cells/well) in cell culture medium and incubated at 37 °C for 6 h. After dying with Cell Tracker Green (Invitrogen, Carlsbad, CA, USA) for 30 min, images of capillary-like structures were captured under a laser scanning confocal microscope (Olympus, Tokyo, Japan). Tubular structures were quantified by manually counting the numbers of connected cells in randomly selected fields at 400× magnification.
Intracellular free calcium imaging
HUVECs were seeded into glass-bottom culture dishes at a density of 2 × 105 cells/dish and were incubated overnight for adhesion. The next day, after incubation with fresh ECM containing Fura-4 AM for 30 min at 37 °C, cells were washed thrice with PBS. All calcium fluorescent images were conducted under an Olympus IX70 microscope with a CCD camera controlled by MetaFluor software (Universal Imaging Corporation, Downingtown, PA, USA). Images were captured every 3 s. After recording the baseline [Ca2+]i for a period of time, both SiNPs at a concentration of 100 μg/mL were added acutely into the dish in the presence or absence of YM-58483 (CRAC inhibitor). The magnitudes of Ca2+ transients induced by SiNPs were represented by the Ca2+ indicator fluorescence intensity and expressed as the ratio of the fluorescence (F/F0) relative to the resting fluorescence (F0).
Non-invasive micro-test to measure transmembrane Ca2+ flux
Non-invasive micro-test (NMT) technology was applied to measure net Ca2+ flux near the plasma membrane of HUVECs using the NMT100 Series (Younger USA LLC, Amherst, MA, USA) equipped with Ca2+-sensitive microelectrodes. This technique allows to non-invasively obtain the dynamic information of ionic or molecular activities on material surfaces . Briefly, HUVECs were treated with the same way as that for intracellular calcium imaging. The Ca2+-sensitive microelectrode was filled with liquid calcium ion-exchanger and calibrated to keep the slop of [Ca2+] change within the standard range. HUVECs were rinsed with external solution composed of (in mmol/L) 120 NaCl, 3 KCl, 2.5 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 25 NaHCO3 and 10 glucose. The microelectrode was positioned to 5 μm near the cell surface and then was moved slightly back and forth controlled by the software. Ca2+ flux data were acquired at a sampling rate of 1 point per 6 s. After 3 min baseline recording, SiNPs of either size at a concentration of 100 μg/mL was acutely added to the dish, and the effects of SiNPs on Ca2+ flux were recorded real-timely and were analyzed using the software. Blank control (without cell) and positive control (high K+, 60 mmol/L) experiments of NMT were also performed in HUVECs.
Dosimetry modeling in vitro
The sedimentation levels of SiNPs onto the cell surface in vitro, which indicate the “effective exposure concentration”, was computationally estimated by the in vitro sedimentation, diffusion and dosimetry (ISDD) model, following treatment with either SiNP-20 or SiNP-100 [27, 47]. The model is available as Matlab code from its developers , and can be downloaded from http://nanodose.pnnl.gov/ModelDownload.aspx. Further details and modeling parameters can be found in the legend of Figure S2.
Animals and in vivo experiments
Female Balb/c mice (8 weeks old, 20–22 g body weight) were purchased from the Experimental Animal Center of Shanxi Medical University (Taiyuan, China). Animals were fed with regular chow and water ad libitum and maintained in 12:12 h light/dark luminosity cycles. Animal experiments were performed to examine the potential hazardous effects of SiNPs on organs in vivo. SiNPs were diluted in 200 μl saline and intravenously (i.v.) injected at 7, 21, and 35 mg/kg (body weight) [21,22,23]. Same volume saline injection was used as controls. Animals were randomly divided to three groups according to treatment: SiNP-20, SiNP-100, and control (n = 6 in each group). Mice were painlessly sacrificed 72 h after injection, organ tissues, including heart, abdominal aorta, lung, liver and kidney, were harvested and morphology changes were examined.
Histochemistry and immunohistochemistry
The tissue samples of heart, aorta, lung, liver and kidney were fixed in 10% formalin, embedded in paraffin, sectioned (5 μm) and attached to slides, deparaffinized, and stained with hematoxylin and eosin (H&E) to generally check the tissue structural changes after exposure to SiNPs. To perform immunohistochemical staining of F4/80 (a macrophage marker) in organ tissues after exposure to SiNPs in vivo, tissue sections were reacted with a 3% hydrogen peroxide/methanol solution to inactivate endogenous peroxidase, washed with PBS, and treated with antigen-unmasking reagent. Tissue sections were then blocked with 10% normal goat serum for 10 min and then incubated with the primary antibody against F4/80 (dilution 1:50) (Abcam, Cambridge, UK) for overnight at 4 °C. The sections were washed with PBS and incubated with avidin-biotin conjugated secondary antibody for 30 min at room temperature, then washed with PBS and reacted with DAB substrate followed by wash with water. Immunoreactive signals in the sections were shot under a microscope.
Immunofluorescent staining was used to examine the effects of SiNPs on the expression and localization of junctional protein VE-cadherin and cytoskeleton protein F-actin in HUVECs in vitro and VE-cadherin in tissues in vivo.
To perform cell staining, HUVECs were seeded into glass-bottom culture dishes at a density of 2 × 105 cells/dish and were incubated overnight for adhesion. Cells were then incubated with both SiNPs at 100 μg/mL in 1 mL culture medium for 2 h. Control cells were treated with same volume of PBS instead of SiNPs. Cells were then fixed with 4% paraformaldehyde for 10 min at 37 °C. After rinsing with fresh PBS, cells were incubated with goat serum for 1 h at room temperature. Cells were then labeled with VE-cadherin XPTM rabbit antibody (dilution 1:200) (Cell Signaling Technology, Danvers, MA, USA) in goat serum at 4 °C for overnight. Thereafter, cells were washed three times with PBS and incubated for 1 h at room temperature with Alexa Fluor®488-labeled goat anti-rabbit antibody (dilution 1:400) (Invitrogen, Carlsbad, CA, USA). F-actin was stained with TRITC-phalloidin (Shanghai Solarbio Bioscience & Technology Co., Ltd., Shanghai, China) additionally. Cells were washed with PBS and mounted with an aqueous mounting medium containing DAPI (Zhongshan Golden bridge biotechnology Co., Beijing, China). Cell fluorescent images were taken using the FluoView FV1000 confocal microscope (Olympus, Tokyo, Japan). The positive fluorescent signals were analyzed using FluoView software (Olympus, Tokyo, Japan).
To perform tissue immunofluorescent staining, tissues were routinely fixed with 10% formalin, dehydrated and embedded in paraffin. Five μm sections were cut with a microtome and mounted on slides, then were deparaffinized and rehydrated and treated with antigen-unmasking reagent. Sections were blocked with 10% normal goat serum for 10 min and then incubated with VE-cadherin XPTM rabbit antibody (dilution 1:200) (Cell Signaling Technology, Danvers, MA, USA) for overnight at 4 °C. The following procedures were the same as that described above in cell immunofluorescent staining. Positive immunofluorescent signals in the tissue sections were captured under a fluorescence microscope.
Western blotting was used to detect the expression levels of VE-cadherin and phosphorylated VE-cadherin (pY731-VEC) in HUVECs after exposure to SiNPs for 6 h. Proteins were quantified with bicinchoninic acid assay (Pierce, Rockford, IL, USA). Equal amounts of proteins were loaded onto a sodium dodecyl sulfate (SDS)-polyacrylamide gel and electrophoretically transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA), then 5% nonfat milk in tris-buffered saline (TBS) was applied to block the membrane for 1 h. The membrane was incubated with the primary anti-VE-cadherin XPTM antibody (dilution 1:1000) or anti-pY731-VEC antibody (dilution 1:500) for overnight at 4 °C. The membrane was then washed thrice with TBS and Tween 20 (TBST) and incubated with a horseradish peroxidase-conjugated anti-rabbit immunoglobulin G secondary antibody (Zhongshan Golden bridge biotechnology Co., Beijing, China) for 1 h at room temperature. After washing thrice with TBST, the positive target signals were detected using enhanced chemiluminescence substrate (Boster Biological Technology, Wuhan, China). Analysis of the protein bands was performed using ImageLab™ Software (Bio-Rad, Hercules, CA, USA).
Data were presented as mean ± standard deviation (SD). Statistical differences between variant treatments were analyzed with the independent sample-test using IBM SPSS Statistic 19 software. The normal distribution of data was tested prior to performing the t-test. Multiple group comparison was performed using analysis of variance. Differences were considered significant at p < 0.05.