Silica NanoparticleâEndothelial Interaction: Uptake and Effect on

Article Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Silica Nanoparticle−Endothelial Interaction: Uptake and Effect on Platelet Adhesion under Flow Conditions Jiban Saikia,†,⊥ Raziye Mohammadpour,† Mostafa Yazdimamaghani,†,§ Hannah Northrup,∥ Vladimir Hlady,*,†,∥ and Hamidreza Ghandehari*,†,§,∥ †

Utah Center for Nanomedicine, Nano Institute of Utah, §Department of Pharmaceutics and Pharmaceutical Chemistry, and Department of Bioengineering, University of Utah, Salt Lake City, Utah 84112, United States ⊥ Department of Chemistry, Dibrugarh University, Dibrugarh, Assam 786004, India ACS Appl. Bio Mater. Downloaded from pubs.acs.org by UNIV OF TEXAS AT EL PASO on 11/07/18. For personal use only.

∥

S Supporting Information *

ABSTRACT: Silica nanoparticles are extensively used in biomedical applications and consumer products. Little is known about the interaction of these NPs with the endothelium and effect on platelet adhesion under flow conditions in circulation. In this study, we investigated the effect of silica nanoparticles on the endothelium and its inflammation, and subsequent adhesion of flowing platelets in vitro. Platelet counts adhered onto the surface of endothelial cells in the presence of nanoparticles increased at both low and high concentrations of nanoparticles. Preincubation of endothelial cells with nanoparticles also increased platelet adhesion. Interestingly, platelet adhesion onto TNF-α-treated endothelial cells decreased in the presence of nanoparticles at different concentrations as compared with the absence of nanoparticles. We monitored the expression of different endothelial proteins, known to initiate platelet adhesion, in the presence and absence of silica nanoparticles. We found that silica nanoparticles caused changes in the endothelium such as overexpression of PECAM that promoted platelet adhesion to the endothelial cell. KEYWORDS: BAEC, endothelium, blood platelets, adhesion, silica nanoparticles, inflammation

1. INTRODUCTION

adhesion leads to recruitment of other platelets, initiating a coagulation cascade that may result in thrombus formation. Silica nanoparticles have been investigated in biomedical research such as in drug delivery applications and as theranostic agents.7,8 When nanoparticles are in the bloodstream, their presence in circulation can cause interactions with the endothelial cell lining, potentially resulting in unintended vascular injury. Nanoparticles are known to interact with the endothelium and cause endothelial dysfunction and inflammation.9,10 Nanoparticles have also been shown to disrupt the NO/NOS cycle and induce oxidative stress and induce expression of different inflammatory markers such as VCAM1, ICAM-1, and PECAM-1.10 Such injury also results in overexpression of a multimeric protein called von Willebrand

The endothelial cell layer (endothelium) separates the circulating blood from surrounding tissues and acts as a biological barrier. The endothelium maintains and regulates hemostasis and thrombosis1,2 and plays a vital role in extravasation of fluids, hormones, and macromolecules.3 Due to its position at the interface between blood and tissues, the endothelium is the first line of defense in the case of vascular injury. Under normal physiological conditions, blood flow is marginated so that the platelets flow in close proximity to vascular walls. Healthy endothelium has an anticoagulant role in maintaining the balance between pro- and anticoagulant events.4 Vascular injury may lead to the denudation of the endothelial layer that results in exposing the subendothelial matrix and also inflammation of endothelial cells. In response to such injury, a coordinated series of events initiate the adhesion of the platelets to the vascular wall.5,6 The platelets © XXXX American Chemical Society

Received: August 26, 2018 Accepted: October 30, 2018 Published: October 30, 2018 A

DOI: 10.1021/acsabm.8b00466 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

synthesized by an addition reaction between 4.3 M (3-aminopropyl) triethoxysilane (APTS, ≥99%, Sigma-Aldrich, St. Louis, MO) and 1.5 mg of TRITC in 10 mL of absolute ethanol (200 proof, Decon Laboratories, Inc. King of Prussia, PA, USA). The mixture was kept under anhydrous nitrogen and a dark environment for 30 min to allow the conjugation of APTS silica precursor with the dye. Next, 0.06 M tetraethoxysilane (TEOS, ≥99%, Sigma-Aldrich, St. Louis, MO) was introduced to the dye precursor solution. The mixture was added dropwise to the reaction vessel containing 5.5 mL of ammonia hydroxide (NH4OH, 28−30% as NH3, EMD Millipore Corporation, Billerica, MA, USA), 3.2 mL of distilled deionized (DI) water, and 90 mL of ethanol and reacted overnight. Subsequently, the cores were coated with 0.11 M TEOS, which was added at a rate of 6.7 μL/min using a syringe pump. The reaction was allowed to take place for 24 h, and the product was centrifuged and washed at 27 000 RCF for 20 min (three times), and the supernatant was replaced with ethanol, it was stored in ethanol.22,23 Size and morphology of the nanoparticles were probed by transmission electron microscopy on a JEOL JEM 1400 microscope (JEOL Ltd., Tokyo, Japan) operating at 120 kV. Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, U.K.) was used to determine ζ potential of the particles. Their fluorescence was measured at excitation/emission wavelengths of 555/575 nm using the SpectraMax M2 (Molecular Devices, Sunnyvale, CA) microplate reader. 2.2. Endothelial Cell Culture and Cell Viability Assay. Bovine aortic endothelial cells (BAECs) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS and grown to confluence under static conditions at 37 °C, with a 5% CO2 atmosphere. For flow experiments, cells were allowed to grow in 35 mm Petri dishes precoated with collagen (Stem cell technologies, Vancouver, Canada) for 45 min. BAECs were seeded at 1 × 106 cells per mL and allowed to grow overnight. To initiate activation and inflammation, the endothelial monolayers were treated with TNF-α (ibidi, Madison, Wisconsin, USA) at 10 ng/mL and allowed to incubate for 12 h. Lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO) applied at 5 μg/mL was used as a positive control to induce inflammation in the endothelial cells. For the viability assay, cells (4 × 103 per well) were seeded in 96-well microliter plates for 24 h before treatment. Cells were exposed to freshly prepared concentrations of nanoparticles in media (3−500 μg/mL) and incubated for 24 h. The medium was replaced with 100 μL of medium solution containing 10% (v/v) CCK-8 reagent. The absorbance of the supernatant at 450 nm was obtained 1−2 h after incubation by scanning with a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA). Each experiment was performed at least three times in triplicate. Cells treated with complete medium involving no particles were used as a negative control and cells treated with 0.01% (v/v) Triton X-100 were used as a positive control. The significant difference between values was considered at the level of p < 0.05. 2.3. Platelet Isolation. Whole human blood was freshly drawn from healthy human donors under protocols approved by the University of Utah Institutional Review Board. Blood was collected into buffered 3.2% (0.105 M) sodium citrate. To prevent thrombininduced coagulation during flow experiments, cells were treated with Phe-Pro-Arg-chloromethylketone (PPACK, 80 lM, Haematologic Technologies, Vermont, USA) within 5 min of blood drawing. The blood was centrifuged at 1500 rpm for 10 min, and the supernatant, platelet rich plasma (PRP), was carefully separated into a new conical tube. The platelets were manually counted using a hemocytometer. Because different batches of blood might have different platelet counts, the PRP suspension was diluted in Tyrode-HEPES buffer (pH 7.5) to ∼2.5 × 107 cells/mL throughout the study. Prior to dilution, the platelets were labeled using DiOC6 (3,3′-dihexyloxacarbocyanine iodide) dye (λex ∼ 485 nm/λem ∼ 501 nm).24 The concentration of the dye for labeling the platelets was optimized to get a better contrast of the platelets while avoiding the dyeing of the endothelial cells during flow experiments. 2.4. Flow Setup and Adhesion Assays of Platelets. The Glycotech flow chamber (model no. 31-001, Glycotech, Gaithersburg, MA) was used in all flow experiments. To flow platelets or

factor (vWF), which is contained in Weibel Palade bodies and synthesized by endothelial cells.11 The overexpression of these proteins is a key initial step in platelet activation and adhesion to the site of injury caused by nanoparticles. For instance, single-walled carbon nanotubes have been shown to induce oxidative stress, which alter the VCAM-1 and ICAM-1 expressions in aortic endothelial cells.12 Similarly exposure of aortic endothelial cells to yttrium oxide (Y2O3) or zinc oxide (ZnO) nanoparticles significantly increases the mRNA expression of the inflammatory marker ICAM-1.10 Titanium oxide nanoparticles have shown to increase induction of mRNA and protein levels of VCAM-1.13 Metal aluminum nanoparticles lead to the increased mRNA and protein expression of ICAM-1 and VCAM-1, in human endothelial cells.14 Nanoparticles may also interact with the intact endothelium and induce transient platelet interactions, which can become an important step in the commencement and progression of atherosclerosis.15,16 In our previous investigations, we found that the shape, size, surface charge, and porosity of silica nanoparticles influence cellular uptake and toxicity.17−20 However, little is known about the influence of silica nanoparticles on endothelial interaction, uptake, and effect on platelet adhesion under flow conditions. The objective of the present study was to examine the role of silica nanoparticles in the induction of expression of endothelial receptors and their association to platelet adhesion with endothelial cells. Platelet adhesion was studied on bovine aortic endothelial cell (BAEC) monolayers in the presence and absence of silica nanoparticles. Endothelial cells were preincubated with the nanoparticles to induce inflammation, and platelet adhesion was studied post exposure of cells to nanoparticles. Silica nanoparticles with a fluorescent core containing rhodamine dye were selected for three reasons: to track the particle interactions with cells in flow experiments, to maintain a constant surface property that would otherwise vary if the fluorescent labels were linked to the particle surface, and to prevent photobleaching of the fluorescent dye now hidden in the particle core.

2. MATERIALS AND METHODS 2.1. Synthesis and Characterization of Dye-Labeled Core/ Shell Structured Silica Nanoparticles (SiNPs). A core/shell structure of silica nanoparticles was obtained via a modified Stöber synthesis,20,21 encapsulating tetramethylrhodamine-5-isothiocyanate (TRITC, Molecular Probes, Eugene, OR) dye (λex ∼ 555 nm/λem ∼ 580 nm) in the core. Mechanism of core/shell TRITC-labeled silica nanoparticles synthesis is depicted in Scheme 1. First, the core was

Scheme 1. Mechanism of Formation of Core/Shell TRITCLabeled Silica Nanoparticles

B


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Figure 1. (A−D) TEM images of core/shell TRITC-labeled silica nanoparticles with different magnifications. The images were used to measure the mean size and polydispersity of nanoparticles. nanoparticles over the endothelial cell monolayer, a flow rate of 0.66 mL/min flow rate was maintained using a syringe pump (Kent Scientific, Torrington, CT). The gasket between the flow chamber and the Petri dish substrate with endothelial cells was selected to produce a shear stress of ∼1 dyn/cm2, mimicking thus a venous blood flow with a wall shear rate of 204 s−1. All microfluidic tubing surfaces were perfused with human serum albumin (HSA, Sigma-Aldrich) before running experiments in order to reduce the adsorption of other serum proteins and platelet activation. Labeled platelets were flowed through the flow chamber over BAECs for 5 min. In some experiments SiNPs of different concentrations were added to the platelets and flowed through the flow chamber. In other sets of experiments, endothelial cells were preincubated overnight with SiNPs, washed with HEPES-Tyrode buffer, and then used in platelet flow experiments. After running platelets, endothelial cells were washed with HEPES-Tyrode buffer for 2 min to remove the unadhered platelets. The attached platelets were imaged using a fluorescence microscope (Diaphot 300, Nikon, with 40× NA 0.55, ELWD objective). At least 5 fluorescence images were recorded from random areas of the BAECs monolayer for each experiment, and each experiment consisted of at least 3 repeats. The number of adhered platelets was counted using ImageJ (NIH, Bethesda, USA) as previously described.25 2.5. Quantitative Real-Time Polymerase Chain Reaction (PCR) Analysis. Transcription levels of four cell adhesion markers, intracellular cell adhesion molecule-1 (ICAM-1) CD54, platelet endothelial cell adhesion molecule-1 (PECAM-1)/CD31, van Willebrand factor (VWF), and P-selectin, were probed. Primers for these markers were obtained from Qiagen (USA). Total RNA was extracted from treated cells using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany). First-strand cDNA was generated by RevertAidTM M-MuLV reverse transcriptase enzyme using 1 μg of total RNA and random primers, with the following program: 25 °C for 10 min and 1 h at 42 °C. Real-time RT-PCR was performed using the QuantiFast SYBR Green PCR Master Mix according to this program: 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, TM for 25 s, and 72 °C for 30 s in a GeneAmp 7900HT Sequence Detection System (Applied Biosystems, Foste City, CA, USA). The data

generated were analyzed, using the comparative CT method, and normalized compared to the bovine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. 2.6. Quantitative Determination of Total Nitric Oxide (NO) in Culture Supernatants. For NO release from the eNOS, we used a total NO concentration determining kit (Enzo Life Science, Inc.; NY, USA) that determines nitric oxide concentration from its two stable breakdown products, nitrate and nitrite, colorimetrically by Griess reaction. The nitrate reductase enzyme was used to convert to nitrite, and this is measured as a colored azo dye product at 540−570 nm.26 For imaging overexpressed protein upon ECs exposure to SiNPs, the particles without the TRITC core but of the same size, d = ∼250 nm, were used. 2.7. Statistical Analysis. The experiments were performed in triplicate, and the results were presented as mean value ± standard deviation. One-way analysis of variance (ANOVA) was performed between treated samples vs controls, unless stated otherwise. The significant difference between values was considered at the level of p < 0.05.

3. RESULTS The goal of the current study was to examine the effects that SiNPs exert on endothelial cells and their interaction with blood platelets. For that purpose, SiNPs with an inner core containing fluorescent dye covered with a silicon oxide shell were synthesized and characterized. TEM images of tetramethylrhodamine-5-isothiocyanate (TRITC)-labeled core/shell nanoparticles revealed uniform particles with a narrow size distribution of 245 ± 10.82 nm in diameter (Figure 1). The ζ potential of SiNPs in water was found to be −52.4 ± 0.6 mV. The fluorescence spectra (excitation 557 nm, emission 576 nm) confirmed the incorporation of the dye into the SiNPs (Figure S1). Based on cell cytotoxicity data (Figure S2), the maximum safe concentration of silica NPs was 250 μg/mL. Two C


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Figure 2. Endothelial cell (EC) monolayer used for the flow experiments [left panel, phase contrast] and image of fluorescent platelets (λex ∼ 485 nm/λem ∼ 501 nm) adhered to EC monolayer [right] panel. Scale bar = 20 μm. Image shown is for the SiNP 50 μg/mL concentration after incubation for 12 h.

concentrations of SiNPs, 10 μg/mL as a low concentration and 250 μg/mL as a high concentration, were selected and used in the mixture with PRP to flow over the endothelial cells. Figure 2 shows representative images of BAEC before (left panel, phase contrast) and after (right panel, fluorescence) the exposure to platelet and SiNP suspension. The adhesion of platelets to an endothelial cell monolayer in the presence of the nanoparticles is shown in Figure 3. The

expression of adhesion molecules.29 These and other literature reports demonstrate that TNF-α is a suitable stimulus for endothelial cell activation.28−30 After the incubation of BAECs with TNF-α, platelet adhesion was found to increase compared to the nontreated control (Figure S5). However, by adding SiNPs to PRP, the adhesion of platelets to the endothelial cells decreased in a concentration-dependent manner (Figure 4). In the presence

Figure 3. Platelet adhesion onto endothelial cells in the presence of different concentrations of SiNPs (**p < 0.01 for platelet adhesion with 10 μg/mL SiNP and *p < 0.10 for platelet adhesion with 250 μg/mL SiNP). (Calculations were added in the Supporting Information, Figures S3 and S4.)

Figure 4. Platelet adhesion onto inflamed endothelial cells (TNF-α treated) in the presence of SiNPs at different concentrations. (Calculations were added in the Supporting Information, Figure S10.)

of 10 μg/mL of SiNPs, platelet adhesion was lower than on TNF-α treated BAECs (i.e., not exposed to nanoparticles). The effect was further amplified at a high concentration, 250 μg/m, of SiNPs (Figures S6 and S7). To elucidate whether BAEC exposure to SiNPs initiates the inflammatory phenotype of the endothelial cells that will affect the extent of platelet adhesion, the endothelial monolayers were first exposed to SiNPs (at 50 μg/mL) for 2 or 12 h under static conditions and used in platelet flow experiments. This SiNP concentration was well below the highest safe concentration (250 μg/mL) of SiNPs. After preincubation, the cells were briefly washed with HEPES-Tyrode buffer and platelet adhesion was measured under flow conditions. Fluorescent images of SiNPs uptake after 2 and 12 h are provided in the supplementary section (Figure S8). Figure 5 shows platelet adhesion after preincubation of the cells with SiNPs at 50 μg/mL. The increase of the preincubation time resulted in a significant increase in platelet adhesion.

number of platelets adhered onto the surface of endothelial cells in the presence of nanoparticles increased at both low and high concentrations of SiNPs. Since the adhesion of the platelets from different blood batches might have differed due to the donor variability, each data set was performed using the same donor blood normalized with respect to the control for each experiment. The calculations and data for obtaining Figure 3 have been provided in the Supporting Information (Figures S3 and S4). To examine the effect of different endothelial cell surface receptors, the BAECs were activated using TNF-α by incubating cells with 10 ng/mL TNF-α for 12 h. TNF-α is known to induce inflammation and overexpression of certain surface receptors on endothelial cells.27 TNF-α increases the expression of the adhesion molecules ICAM-1, VCAM-1 and E-selectin on the surface of vascular endothelial cells.28 A concentration of 10 ng/mL is reported to modulate the D


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4. DISCUSSION Endothelial cell exposure to silica nanoparticles could be intentional (e.g., in food additives and biomedical products) or unintentional absorption into circulation (by environmental exposure). Such exposure may result in the inflammation of the endothelium or even in vascular injury. The inflamed endothelium may in turn express different proteins that will induce platelet adhesion and activation. To examine the interactions between endothelial monolayers and silica nanoparticles, fluorescent core−shell silica nanoparticles were used to enable particle visualization during interaction with the endothelial cells. Regardless of the platelet activation state, both resting and activated platelets adhere primarily to subendothelial matrix proteins, rather than to endothelial cells.32 However, platelets are also known to bind to adhesive proteins expressed by activated endothelial cells. An upsurge in the number of platelets adhered onto the surface of BAECs in the presence of nanoparticles was observed as compared to the control (no nanoparticles) (Figure 3). The presence of nanoparticles in PRP is not expected to increase the number of transient platelet−BAECs contacts. It is therefore likely that the nanoparticles themselves triggered overexpression of endothelial cell receptors, to which platelets adhered. One would expect that the higher concentration of SiNPs in PRP could also hinder platelet interaction with the endothelial cell surface causing a reduction in platelet adhesion (Figure 3). This could occur due to a competitive binding between platelets and nanoparticles to expressed endothelial adhesion and signaling molecules, since it was previously reported that nanoparticles specifically accumulate in areas of vascular injury.33 Preincubation of endothelial cells with nanoparticles also increased platelet adhesion (Figure 4). It is possible that nanoparticles are interacting with platelets, especially if the nanoparticles are coated in plasma with proteins that can act as platelet agonists. However, such nanoparticle-primed platelets will still need specific receptors to bind to endothelial cell surface. These receptors should not

Figure 5. Platelet adhesion onto endothelial cells pretreated with 50 μg/mL SiNPs for 2 h (*p < 0.05 for platelet adhesion with 50 μg/mL SiNP for 12 h). (Calculations were added in the Supporting Information, Figure S10.)

Calculation and representative images are included in the supplementary section (Figures S9 and S10). Transcription levels of cell adhesion markers PECAM-1, ICAM-1, vWF, and P-selectin on an endothelial surface were probed to examine which one is involved in the inflammatory response of BAECs upon interaction with SiNPs. SiNPs at 50 μg/mL were incubated for 2 or 12 h with BAECs and the mRNA expression levels were measured relative to nontreated control cells. TNF-α- and LPS-treated endothelial cells were used as positive controls of the inflammatory response.31 Figure 6 and Table 1 show the results of mRNA expression profile genes. Endothelial NO synthase (eNOS) is one of the NOS isoforms that is present specifically in endothelial cells. Figure 7 quantifies the amount of NO from the endothelial cells in the presence and absence of SiNPs (50 μg/mL). The positive control included cells treated with TNF-α or LPS. The release of nitric oxide upon treatment with SiNPs was found to be greater than the control.

Figure 6. mRNA expression levels of cell adhesion markers responsible for platelet adhesion and inflammation (*p < 0.05, **p < 0.01, and ***p < 0.001). E


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Table 1. Gene Expression Value of ICAM, PECAM, P-selectin, and VWF in Treated Cells Normalized to Non-Treated Cells ICAM control LPS SI-12H SI-2H TNF

1 9.571 −1.3 −3.48 7.45

± ± ± ± ±

0.0014 0.248 0.054 0.032 0.184

PCAM 1 −2.34 2.03 −1.82 −2.27

± ± ± ± ±

P-selectin

0.0016 0.0069 0.0084 0.0014 0.002

1 24.98 −2.058 −1.44 17.21

± ± ± ± ±

0.0082 0.472 0.0048 0.007 0.227

VWF 1 2.31 −1.163 −2.55 3.95

± ± ± ± ±

0.024 0.0513 0.004 0.0002 0.048

exposure to SiNPs did not result in an increase of the expression of PECAM-1 genes. Nanoparticles can also disturb the NO/NOS system by inducing reactive oxygen species generation, altering activities of NOS and eNOS significantly and inducing increased NOS (iNOS).41 The uptake of nanoparticles via the caveolaemediated mechanism also induces NO production.42 NO is an effective vasodilator and an inhibitor of leukocyte and platelet adhesion.43 Such effects are mediated by activation of soluble guanylate cyclase, resulting in an increased intracellular cyclic guanosine monophosphate (GMP). The role of NO as an inhibitor of platelet adhesion has been linked to both dependent and independent cyclic GMP mechanisms.44 One of the key determinants of platelet adhesion is the balance between reactive oxygen species and NO. In the present study, the amount of NO was measured using a total NO detection kit.45 As expected, NO release was compromised when the endothelial cells were inflamed (Figure 7). However, the release of NO upon 12 h treatment with SiNPs was found to be greater than the controls. Although the addition and uptake of SiNPs might cause an alteration in the endothelial monolayer, the observed increase of NO might be due to other factors and not limited to the endothelial injury. It has been reported that aortic endothelial cells can uptake nanoparticles via caveolae-mediated endocytosis, which activates eNOS and in turn increases NO production.42 The increased production of NO observed here might be due to the same reason. The increase in the number of platelets in our current studies is in contrast to a previous report examining smaller mesoporous silica nanoparticles.42 In that report, the investigators used 50 nm mesoporous SiNPs in the concentration range of ∼20 and 200 μg/mL and found a decrease in platelet adhesion compared to controls and compared to a higher concentration of ∼1000 μg/mL. Such inconsistency may be attributed to the difference in particle size (∼50 nm used in previous study compared to ∼250 nm in the present study) and porosity (mesoporous used in the previous study vs Stöber SiNP particles used in the current study). We have previously shown that the size and porosity clearly influence cytotoxicity in vitro and maximum tolerated dose in vivo.18,20,46,47 These observations underline the importance of a careful examination of structural properties of SiNPs, such as size, size distribution, surface charge, porosity, and geometry on silica nanoparticle−endothelial interactions, uptake and effect on platelet adhesion under flow conditions.

Figure 7. Nitric oxide released by BAECs under different conditions (*p < 0.05 and **p < 0.01).

be present on nonactivated endothelial cells. Interestingly, platelet adhesion onto TNF-α-treated endothelial cells decreased in the presence of nanoparticles at different concentrations as compared with the absence of nanoparticles (Figure 5). Nanoparticles are reported to interact directly with an endothelial monolayer and induce endothelial system inflammation and dysfunction.34 They were found to induce both mRNA as well as protein expression of endothelial adhesion molecules (ICAM-1).35 Platelet endothelial cell adhesion molecule-1 (PECAM-1)/CD31, intercellular adhesion molecule-1 (ICAM-1)/CD54, vWF, and P-selectin are proteins expressed on the surface of endothelial cells that could be directly or indirectly responsible for platelet adhesion and subsequent activation. ICAM-1 has been found to mediate platelet adhesion to the endothelial surface via a bridging mechanism that involves platelet-bound adhesive proteins.32 Endothelial cells also sequester vWF protein and keep it within their storage granules, known as Weibel−Palade bodies (WPB) to strengthen the interaction of platelets with the basement membrane, by keeping it within their storage granules, known as Weibel−Palade bodies.36 Glycoprotein Ibα (GIbα) on the platelet membrane interacts with the vWF initiating platelet adhesion at high shear rates (>1000 s−1).37 Inflammation and activation of the endothelial cells also release another protein from the storage granule (Weibel−Palade bodies) known as Pselectin. P-selectin exposure to the surface of the inflamed endothelial cells may lead to the interaction via the platelet GPIb complex. Endothelial cell P-selectin also mediates the initial transient interactions of leukocytes with endothelial cells (i.e., rolling), which is associated with platelet adhesion.38 The present study showed that only PECAM-1 gene expression increased after 12 h exposure of BAECs to SiNPs (Figure 6). PECAM-1/CD31 is an endothelial cell protein that has been reported to play a significant role in modulating platelet adhesion/aggregation at the site of minor endothelial injury. Although the endothelial cells are intact in minor injury, PECAM may be expressed to promote platelet adhesion and subsequent aggregation.39,40 Interestingly, a shorter 2 h

5. CONCLUSION The objectives of this study were to investigate the role of the Stöber silica nanoparticle of approximately 250 nm in diameter on the interaction with the endothelial cells and to study the expression of endothelial cell surface receptors implicated in the platelet adhesion interaction. It was found that in the F


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(3) Mehta, D.; Malik, A. B. Signaling Mechanisms Regulating Endothelial Permeability. Physiol. Rev. 2006, 86, 279−367. (4) Rumbaut, R. E.; Thiagarajan, P. Platelet-Vessel Wall Interactions in Hemostasis and Thrombosis. In Synthesis Lectures on Integrated Systems Physiology: From Molecule to Function; Morgan & Claypool Life Sciences, San Rafael, CA: 2010; Vol. 2, pp 1−75. (5) Fujimura, Y.; Titani, K.; Holland, L.; Russell, S.; Roberts, J.; Elder, J.; Ruggeri, Z.; Zimmerman, T. von Willebrand Factor. A Reduced and Alkylated 52/48-kDa Fragment Beginning at Amino Acid Residue 449 Contains the Domain Interacting with Platelet Glycoprotein Ib. J. Biol. Chem. 1986, 261, 381−385. (6) Varga-Szabo, D.; Pleines, I.; Nieswandt, B. Cell Adhesion Mechanisms in Platelets. Arterioscler., Thromb., Vasc. Biol. 2008, 28, 403−412. (7) Rühle, B.; Saint-Cricq, P.; Zink, J. I. Externally Controlled Nanomachines on Mesoporous Silica Nanoparticles for Biomedical Applications. ChemPhysChem 2016, 17, 1769−1779. (8) Chen, F.; Hableel, G.; Zhao, E. R.; Jokerst, J. V. Multifunctional Nanomedicine with Silica: Role of Silica in Nanoparticles for Theranostic, Imaging, and Drug Monitoring. J. Colloid Interface Sci. 2018, 521, 261−279. (9) Engin, A. B. Nanoparticle: As a messenger between environment and endothelium. In Endothelium: Molecular Aspects of Metabolic Disorders; Engin, A. B., Engin, A., Eds.; CRC Press/Taylor & Francis Group: 2013; pp 428−449. (10) Gojova, A.; Guo, B.; Kota, R. S.; Rutledge, J. C.; Kennedy, I. M.; Barakat, A. I. Induction of Inflammation in Vascular Endothelial Cells by Metal Oxide Nanoparticles: Effect of Particle Composition. Environ. Health Perspect. 2007, 115, 403. (11) Ruggeri, Z. M. The Role of von Willebrand Factor in Thrombus Formation. Thromb. Res. 2007, 120, S5−S9. (12) Zhiqing, L.; Zhuge, X.; Fuhuan, C.; Danfeng, Y.; Huashan, Z.; Bencheng, L.; Wei, Z.; Huanliang, L.; Xin, S. ICAM-1 and VCAM-1 Expression in Rat aortic Endothelial Cells after Single-Walled Carbon Nanotube Exposure. J. Nanosci. Nanotechnol. 2010, 10, 8562−8574. (13) Han, S. G.; Newsome, B.; Hennig, B. Titanium Dioxide Nanoparticles Increase Inflammatory Responses in Vascular Endothelial Cells. Toxicology 2013, 306, 1−8. (14) Oesterling, E.; Chopra, N.; Gavalas, V.; Arzuaga, X.; Lim, E. J.; Sultana, R.; Butterfield, D. A.; Bachas, L.; Hennig, B. Alumina Nanoparticles Induce Expression of Endothelial Cell Adhesion Molecules. Toxicol. Lett. 2008, 178, 160−166. (15) Massberg, S.; Brand, K.; Grüner, S.; Page, S.; Müller, E.; Müller, I.; Bergmeier, W.; Richter, T.; Lorenz, M.; Konrad, I.; et al. A Critical Role of Platelet Adhesion in the Initiation of Atherosclerotic Lesion Formation. J. Exp. Med. 2002, 196, 887−896. (16) Huo, Y.; Schober, A.; Forlow, S. B.; Smith, D. F.; Hyman, M. C.; Jung, S.; Littman, D. R.; Weber, C.; Ley, K. Circulating Activated Platelets Exacerbate Atherosclerosis in Mice Deficient in Apolipoprotein E. Nat. Med. 2003, 9, 61. (17) Yazdimamaghani, M.; Moos, P. J.; Ghandehari, H. Global Gene Expression Analysis of Macrophage Response Induced by Nonporous and Porous Silica Nanoparticles. Nanomedicine 2018, 14, 533−545. (18) Yu, T.; Malugin, A.; Ghandehari, H. Impact of Silica Nanoparticle Design on Cellular Toxicity and Hemolytic Activity. ACS Nano 2011, 5, 5717−5728. (19) Herd, H.; Daum, N.; Jones, A. T.; Huwer, H.; Ghandehari, H.; Lehr, C.-M. Nanoparticle Geometry and Surface Orientation Influence Mode of Cellular Uptake. ACS Nano 2013, 7, 1961−1973. (20) Saikia, J.; Yazdimamaghani, M.; Hadipour Moghaddam, S. P.; Ghandehari, H. Differential Protein Adsorption and Cellular Uptake of Silica Nanoparticles Based on Size and Porosity. ACS Appl. Mater. Interfaces 2016, 8, 34820−34832. (21) Stö ber, W.; Fink, A.; Bohn, E. Controlled Growth of Monodisperse Silica Spheres in the Micron Size Range. J. Colloid Interface Sci. 1968, 26, 62−69. (22) Burns, A.; Sengupta, P.; Zedayko, T.; Baird, B.; Wiesner, U. Core/Shell Fluorescent Silica Nanoparticles for Chemical Sensing: Towards Single-Particle Laboratories. Small 2006, 2, 723−726.

presence of these nanoparticles the platelet−endothelial interaction was enhanced. In contrast, for intentionally inflamed (TNF-α-treated) endothelial cells, the same trend was not observed. The gene expression analysis indicates that silica nanoparticles might have an increased platelet adhesion by increasing the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) on the surface of cells.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.8b00466.

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Fluorescence spectra of unlabeled and TRITC corelabeled silica nanoparticles; cell viability assay of BAECs exposed to silica nanoparticles; detailed calculations of all of the figures with representative fluorescence images (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jiban Saikia: 0000-0002-7422-9006 Raziye Mohammadpour: 0000-0002-6155-6518 Mostafa Yazdimamaghani: 0000-0001-5090-9528 Hannah Northrup: 0000-0002-1589-0224 Vladimir Hlady: 0000-0002-7523-6769 Hamidreza Ghandehari: 0000-0002-9333-9964 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Support from the National Institute of Environmental Health Sciences of the NIH (R01 ES024681), the National Heart, Lung and Blood Institute (R01 HL126864), The University of Utah Nanotechnology Training Program (H.N.), and College of Pharmacy Skaggs fellowship (M.Y.) is acknowledged. This work made use of The University of Utah facilities of the Micron Microscopy Suite and The University of Utah USTAR shared facilities supported in part under Award no. DMR1121252 by the MRSEC Program of the National Science Foundation (NSF).

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ABBREVIATIONS PRP, platelet rich plasma; DMEM, Dulbecco’s modified Eagle medium; FBS, fetal bovine serum; SiNP, silica nanoparticle; VCAM-1, vascular cell adhesion molecule 1; PECAM-1, platelet endothelial cell adhesion molecule; vWF, Willebrand factor; PPACK, Phe-Pro-Arg-chloromethylketone; DiOC6, 3,3′-dihexyloxacarbocyanine iodide; PCR, polymerase chain reaction; ICAM-1, intercellular adhesion molecule 1; TRITC, thetramethylrhodamine-5-isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NO/NOS, nitric oxide/ nitric oxide synthesis; BAECs, bovine aortic endothelial cells

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