The effect of protein corona composition on the interaction of carbon nanotubes with human blood platelets

Biomaterials xxx (2014) 1e13

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The effect of protein corona composition on the interaction of carbon nanotubes with human blood platelets Silvia H. De Paoli a, Lukas L. Diduch c, Tseday Z. Tegegn a, Martina Orecna a, Michael B. Strader a, Elena Karnaukhova a, John E. Bonevich c, Karel Holada b, Jan Simak a, * a b c

Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993-0002, USA Institute of Immunology and Microbiology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic National Institute of Standards and Technology, Gaithersburg, MD 20899, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 February 2014 Accepted 16 April 2014 Available online xxx

Carbon nanotubes (CNT) are one of the most promising nanomaterials for use in medicine. The blood biocompatibility of CNT is a critical safety issue. In the bloodstream, proteins bind to CNT through noncovalent interactions to form a protein corona, thereby largely defining the biological properties of the CNT. Here, we characterize the interactions of carboxylated-multiwalled carbon nanotubes (CNTCOOH) with common human proteins and investigate the effect of the different protein coronas on the interaction of CNTCOOH with human blood platelets (PLT). Molecular modeling and different photophysical techniques were employed to characterize the binding of albumin (HSA), fibrinogen (FBG), g-globulins (IgG) and histone H1 (H1) on CNTCOOH. We found that the identity of protein forming the corona greatly affects the outcome of CNTCOOH’s interaction with blood PLT. Bare CNTCOOH-induced PLT aggregation and the release of platelet membrane microparticles (PMP). HSA corona attenuated the PLT aggregating activity of CNTCOOH, while FBG caused the agglomeration of CNTCOOH nanomaterial, thereby diminishing the effect of CNTCOOH on PLT. In contrast, the IgG corona caused PLT fragmentation, and the H1 corona induced a strong PLT aggregation, thus potentiating the release of PMP. Published by Elsevier Ltd.

Keywords: Carbon nanotubes Nanoparticles Platelets Biocompatibility Nanotoxicity Protein corona

1. Introduction Carbon nanotubes (CNT), one of the fundamental platforms of nanotechnology, exhibit a broad range of potential applications, ranging from renewal energy to medicine [1]. Because of their unique mechanical, thermal and electronic properties, CNT are very attractive candidates for use in tissue engineering [2], neuronal growth [3], diagnostic biosensors [4], drug delivery nanosystems [5], imaging nanoprobes for intravascular use [6], and other devices [7] that come into direct contact with blood. It is well recognized that protein adsorption plays an important role in bodily responses to implanted materials and devices, including the modulation of complement activation and blood coagulation [8]. In the case of nanomaterials, once they arrive in the bloodstream or tissue interstitial fluids, different proteins bind to nanoparticle surfaces through non-covalent interactions. This protein coating layer known as the ‘protein corona’ largely defines the biological identity of the nanoparticles [9,10]. The formation of a protein corona has

* Corresponding author. E-mail address: [email protected] (J. Simak).

several consequences. Protein coronas have been shown to influence nanoparticle cellular uptake, trafficking, opsonization, and biodistribution, leading to changes in the circulation time. As such, protein coronas may affect the performance of biomedical nanodevices and modulate the toxicological profiles of nanoparticles [11e13]. We have previously shown that CNT interact with blood platelets (PLT), thereby triggering PLT activation and the release of platelet membrane microparticles (PMP) [14,15]. Other studies have shown that the pre-incubation of CNT with albumin decreases the thrombogenicity [16] and cytotoxicity of CNT [17]. These findings highlight the importance of understanding the formation of protein coronas on nanomaterials to design safer and more efficient nanoparticles for biomedical use. Blood PLT, aside from their primary physiological role in hemostasis [18], have a number of complex non-hemostatic functions. Their ability to expose different receptors and release various bioactive molecules enables them to perform several sentinel tasks and to quickly communicate with the cells of the immune system. PLT play a critical role in host defense, pathogen surveillance, and wound healing and are pivotal mediators of cellular communications during the inflammatory response [18]. Therefore, studies on

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the effects of materials on PLT function are required to demonstrate the biocompatibility of the nanomaterials that come into direct contact with blood. Until now, the importance of protein corona composition on the interaction of CNT with blood PLT has remained unclear. In the plasma, a complex corona composed of hundreds of proteins form around the CNT backbone [9]. Here, we evaluated the formation of the coronas on CNTCOOH using selected individual human proteins. Carboxylic acid functionalized CNT were chosen because this is the most common method to convert insoluble pristine CNT to a water-soluble counterpart. Additionally, carboxylic acid groups allow for a facile bioconjugation of CNTCOOH during the engineering of CNT-based platforms for multicomponent systems [19]. Interestingly, it has been shown that carboxylated nanoparticles attract more proteins from the plasma than do non-functionalized or amine-modified nanoparticles [9]. We studied the coronas formed by the most abundant human plasma proteins: human serum albumin (HSA), fibrinogen (FBG), and gglobulins (IgG). The relevance of our choice was affirmed by onedimensional liquid chromatography tandem mass spectrometry (1D LC-MS/MS) analysis, which confirmed the presence of IgG, HSA and FBG as the major components of the corona formed after CNTCOOH’s exposure to human plasma. In addition, the adsorption of these proteins on foreign materials is known to influence the physiological response and biocompatibility [20,21]. We also included histone H1 in our studies as a representative of highly alkaline proteins that have much higher isoelectric points than HSA, FBG or IgG. Despite their predominant physiologic nuclear localization, histones have been found in the circulation of patients with certain pathologies, where they can be released from dying cells or actively secreted by activated inflammatory cells [22]. 2. Materials and methods 2.1. Materials Tyrode’s buffer (#T2397; without BSA), human fibrinogen (#F4883); human gglobulins (#G4386), human serum albumin (#3782) and calf thymus histone H1 (#H5505) were purchased from SigmaeAldrich; Thrombin Receptor Activating Peptide-6 (TRAP) was from Anaspec; general oxidative stress indicator 5-(and-6)chloromethyl-20 ,70 -dichlorodihydrofluorescein diacetate, acetyl ester (CMH2DCFDA; #C6827), membrane labeling Oregon GreenÒ 488 DHPE (1,2dihexadecanoyl-sn-glycero-3-phosphoethanolamine) (#O-12650), and ProLongÒ Gold Antifade Reagent (#P36930) were from Life Technologies; LDH Cytotoxicity Detection Kit (Takara Bio, Japan) was from Clontech; and the Pierce BCA Protein Assay Kit was from Thermo Scientific (#23225). FITC Mouse Anti-Human CD41a (#555466; clone HIP8), PE Mouse Anti-Human CD62P (#555524; clone AK-4), PE AnnexinV (#556422), PE conjugated Mouse IgG1 (#559320, clone MOPC-21; isotype control) and FITC conjugated Mouse IgG1 (# 555748, clone MOPC-21; isotype control) were from BD Pharmingen. Osmium tetroxide, uranyl acetate and lead citrate were from Ted Pella; EMBed-812 embedding media (#14120) and cacodylate buffer were from Electron Microscopy Science. All of the suspensions were prepared in Bio Plas 4202SLS siliconized microcentrifuge tubes (from Fisher Scientific) to prevent protein adsorption. Centrifugal filters with pore sizes of 0.1 mm (#UFC30VV00; Ultrafree) were from Millipore. The blood of healthy donors (ACD anticoagulated) was obtained from the Department of Transfusion Medicine, Clinical Center, NIH, Bethesda, MD. Platelet-rich plasma (PRP) was isolated from whole blood by centrifugation at 150 g for 10 min at RT, and platelet-poor plasma (PPP) was collected through the centrifugation of PRP at 1000 g for 20 min. 2.2. Preparation of CNTCOOH Multi-walled carbon nanotubes (MWCNT; purity > 95%) with outer diameters of 60e100 nm and lengths of 1 mme2 mm were purchased from SES Research (#9001280). Water-soluble carboxylic acid-modified MWCNT were prepared from pristine MWCNT by stirring in a concentrated 3:1 (v/v) H2SO4 and HNO3 mixture at 70  C for 24 h, followed by extensive washing with deionized water and spin dialysis in centrifugal filter units, with a 3 kDa cutoff membrane (#UFC800324; Millipore) [23]. 2.3. Preparation of protein-coated CNTCOOH Human plasma protein corona or individual protein corona onto CNTCOOH were prepared by incubating 100 mL of CNTCOOH (1 mg/mL in PBS) with 1 mL of plasma (PPP) or an individual protein solution (1 mg/mL) for 1 h [9]. Unbound proteins were

removed by the centrifugation of CNTCOOH at 10,000 g and washing 2 with Tyrode’s buffer. 2.4. LC-MS/MS analysis of plasma protein corona formed on CNTCOOH after incubation with human plasma CNTCOOH bound proteins were denatured and reduced in 6 mol/L urea, 20 mmol/L DTT, and 100 mmol/L NH4HCO3 at a pH of 8.0 at 60  C for 1 h, followed by alkylation with 100 mmol/L iodoacetamide. Samples were then diluted with 2 mol/L urea, 100 mmol/L NH4HCO3, and a pH of 8.0 at 22  C, and each sample was digested overnight with trypsin (1:25 enzyme:substrate ratio) at 37  C. The peptides were lyophilized, desalted using solid-phase extraction, dried down, and resuspended in 0.1% formic acid. The peptides were analyzed using reverse phase one-dimensional liquid chromatography mass spectrometry (1D LC-MS/MS) with an Easy nLC II Proxeon Nanoflow HPLC system that was coupled online to a Q-Exactive Orbitrap mass spectrometer (Thermo Scientific). The survey scans were acquired in the Orbitrap analyzer at a resolution of 70.000, and the fragment ions were acquired with a resolution of 17.000 (see Supplementary information). MS data files were searched against the Swiss-Prot Human database (20270 entries) supplemented with the porcine trypsin sequence using the Mascot search engine (Matrix Sciences; version 2.4.0). The Mascot output files were analyzed using the software Scaffold 4.2.0 (Proteome Software Inc.). Protein identifications were accepted if they could be established at greater than a 99.9% probability and contained at least 2 identified peptides (Table SI 1). Protein probabilities were assigned by the Protein Prophet algorithm. Proteins that contained similar peptides and could not be differentiated based on an MS/MS analysis alone were grouped to satisfy the principles of parsimony. 2.5. Electron microscopy imaging of protein corona formed on CNTCOOH Protein-coated CNTCOOH (Section 2.3) were resuspended in 500 mL of cacodylate buffer. A total of 10 mL of solution was placed on an ultra-thin, carbon-coated grid. To visualize the protein coronas as a dark shadow on TEM, the grids were stained with 1% uranyl acetate [24]. Images were taken using a JEOL 1400 TEM (equipped with a Gatan Orius SC1000 camera) operating with an accelerating voltage of 80 kV. 2.6. Protein conformational assessment by circular dichroism and fluorescence spectroscopy Circular dichroism (CD) spectra were recorded on the Jasco J-815 Spectropolarimeter at 25  0.2  C in a quartz cuvette with a 2 mm path length; the temperature was maintained by a Peltier thermostat. Aliquots of 10 mL and 30 mL of CNTCOOH solution (3 mg/mL in Tyrode’s buffer) were added to the 5 mmol/L protein solution in Tyrode’s buffer. The far-UV CD spectrum of bare CNTCOOH was recorded by adding 40 mL of CNTCOOH solution to 500 mL of the protein-free Tyrode’s buffer. The spectra were measured in triplicate with a scan speed of 100 nm/min, a bandwidth of 1.0 nm, and a resolution of 0.2 nm. The baseline was subtracted using Tyrode’s buffer as a blank. The unsmoothed CD spectra were analyzed for secondary structural elements using the software package CDPro/CONTIN with the following references: SP43 for HSA, IgG and H1, and CLSTR for FBG. Protein conformational changes were studied also by fluorescence spectroscopy (see Supplementary Information). 2.7. Analysis of protein surface properties by PyMOL The structures of FBG, HSA, IgG and H1 were illustrated by PyMOL molecular graphics (version 1.6; http://www.pymol.org) from the Protein Data Bank (PDB) files 3GHG [25], 1AO6 [26], 1HZH [27] and 1GHC [28], respectively. Protein structures rendered by PyMOL were used to determine protein dimensions, surface areas, and the numbers of aromatic (Trp, Tyr, Phe and His) and hydrophobic residues (Phe, Ile, Trp, Leu, and Val) on the protein surface. Surface rendering was achieved by setting the surface-accessible solvent radius to 1.4 Å. The electrostatic potential (distribution of positively and negatively charged domains) on the proteins’ surfaces was calculated using the Adaptive PoissoneBoltzmann Solver (APBS) [29,30] in PyMOL; electrostatic potentials were obtained at a 150 mmol/L ionic strength, with a solute dielectric of 2 and a solvent dielectric of 78.5 [29]. 2.8. Effect of protein corona on agglomeration state of CNTCOOH The effect of protein corona formation on CNTCOOH agglomeration state was measured by light transmission aggregometry using the PAP-8E Platelet Aggregation Profiler from Bio/Data Corporation. In total, 450 mL of 100 mg/mL CNTCOOH in PBS was added to an aggregometry tube and stirred at 150 RPM or 300 RPM. Then, 50 mL of protein solution (10 mg/mL) was added to the tube containing the CNTCOOH solution (protein final concentration 1 mg/mL), and the changes in light transmission due to CNTCOOH agglomeration were measured for 15 min. Bright-field microscopy images of protein-coated CNTCOOH were taken via Zeiss laser scanning confocal microscopy (LSCM) 710 LSM using a Plan Apochromat 63/1.40 oil objective.

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S.H. De Paoli et al. / Biomaterials xxx (2014) 1e13 2.9. Quantification of proteins bound to CNTCOOH BCA protein assay was used to determine the amount of protein bound to CNTCOOH. A total of 1 mL protein solution (1 mg/mL freshly prepared in Tyrode’s buffer) was mixed with 100 mL of CNTCOOH (1 mg/mL in PBS) and agitated gently for 1 h. The solutions were centrifuged at 15,000 g for 10 min, and the remaining protein found in the supernatant corresponded to those proteins that did not interact with the CNTCOOH. BCA analysis of supernatant was performed in accordance with the manufacturer’s instructions, and a minimum of five independent measurements were performed. 2.10. Zeta potential of protein-coated CNTCOOH In total, 1 mL of protein solution (1 mg/mL freshly prepared in Tyrode’s buffer) was mixed with 100 mL of CNTCOOH (1 mg/mL in PBS) and agitated gently for 1 h. The solutions were centrifuged at 15,000 g for 10 min and washed twice with Tyrode’s buffer. The zeta potential (z) of protein-coated CNTCOOH (in 1 mL of Tyrode’s buffer) was measured using a ZetaSizer Nano-ZS (Malvern Instruments) at RT. 2.11. Incubation of CNTCOOH with human blood PLT To obtain washed PLT from the whole blood, PRP containing 1 mmol/L of prostaglandin E1 was centrifuged at 1000 g for 15 min. PLT pellets were carefully resuspended in Tyrode’s buffer without BSA to a final 300  103 PLT/mL suspension. The interactions of CNTCOOH with PLT were analyzed by mixing 0.9 mL of the PLT suspension with 100 mL of CNTCOOH or protein-coated CNTCOOH suspensions (1 mg/mL) or with TRAP (positive control; 20 mmol/L final concentration) [31]. The solutions were incubated at RT for 20 min under gentle agitation. 2.12. Ultra-structural analysis of the interaction of CNTCOOH with human blood PLT The ultra-structural analysis of the protein-coated CNTCOOH interaction with PLT was performed by transmission electron microscopy (TEM). After treatment, the PLT samples were fixed in 4% paraformaldehyde and 1% glutaraldehyde for 1 h and were post-fixed with 4% osmium tetroxide (OsO4) solution in cacodylate buffer 0.1 mol/L. The samples were then dehydrated in an ethanol series, embedded with EMBed-812 and polymerized for 2 days at 65  C. Ultrathin sections (approximately 50 nm) were cut on a Leica ultramicrotome; thin sections were collected onto 600mesh copper grids and stained with uranyl acetate and lead citrate. 2.13. PLT aggregometry To assess the effect of protein-coated CNTCOOH on PLT aggregation, we employed light transmission aggregometry (PAP-8E Platelet Aggregometer Profiler, Bio/Data Corp., Horsham, PA), as previously described [14,15]. Aggregation experiments were completed within 4 h after blood collection to ensure normal PLT responsiveness. Then, 0.9 mL of PLT suspension (300  103 PLT/mL in Tyrode’s buffer without BSA) was placed in aggregometer cuvettes under magnetic stirring, and 100 mL of CNTCOOH or protein-coated CNTCOOH suspensions (1 mg/mL) or of TRAP (positive control; 20 mmol/L final concentration) was added to the PLT suspension. Light transmission measurements were recorded for 20 min. Each experiment was performed using at least 3 blood samples from different donors. The means of the maximum aggregation responses  SD are presented. 2.14. Laser scanning confocal microscopy PLT membranes were labeled using Oregon GreenÒ 488 DHPE. In total, 1 mL of PRP was incubated with 0.1 mmol/L of the label for 15 min at 37  C. A PLT suspension containing 1 mmol/L of prostaglandin E1 was centrifuged at 1000 g for 15 min. PLT pellets were carefully resuspended in Tyrode’s buffer without BSA to a final 300  103 PLT/mL suspension. After treatment with CNTCOOH, PLT were fixed with 2% paraformaldehyde for 30 min at RT and then deposited onto a clean poly-L-lysine coated glass slide by cytospin. Slides were washed 3 times with PBS, mounted with Gold ProLong Antifade and imaged using a Zeiss 710 LSM with a Plan Apochromat 63/1.40 oil objective. Once adjusted, the laser configuration and pinhole aperture were kept the same for all of the samples analyzed. 2.15. Flow cytometry analysis of PLT membrane microparticles (PMP) Aliquots of PLT suspension were incubated with 10 mg/mL protein-coated CNTCOOH (Section 2.11) and centrifuged at 1000 g (5 min, 22  C) to sediment the PLT. The supernatant containing PMP was collected, and the PMP were labeled for flow cytometry, as previously described [15]. Briefly, 50 mL of PMP suspension was incubated for 20 min at RT in the dark with a saturating concentration (5 mL) of monoclonal antibodies against CD41a (FITC) and CD62P (PE) or AnnexinV (PE). Matching IgG isotype controls and non-labeled samples were used as controls. After incubation, the samples were transferred to centrifugal filters (Ultrafree Millipore; 0.1 mm pore size) that contained 800 mL of HBSS (with Ca2þ) and were then centrifuged at 12,000 g for 3 min at 10  C. PMP were resuspended in 500 mL of HBSS (with Ca2þ) and analyzed by BD LSRII flow cytometer using the forward scatter (FSC), and side scatter (SSC) in logarithmic mode. Polystyrene nanobeads 300e 1000 nm (Bang Laboratories, Inc.) were used as size standards, and PMP were

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defined as particles 1000 nm. The flow rate was evaluated using TruCount beads (BD), and counts of CD41aþCD62Pþ, or CD41aþAnnexinVb PMP were evaluated using double fluorescence plots acquired over 60 s at the standard flow rate. 2.16. PLT Oxidative stress (ROS) induced by CNTCOOH A cell-permeant indicator for reactive oxygen species in living cells (CMH2DCFDA) was used to measure the ROS production in PLT. PLT in PRP were incubated with 6 mmol/L H2DCF-DA for 30 min at 37  C. Any excess dye was removed by pelleting the PLT at 1000 g for 15 min. The PLT were then resuspended in Tyrode’s buffer at 300  103 PLT/mL, and 450 mL aliquots were transferred to 24-well plates. The ROS production kinetics was measured after the addition of 50 mL of proteincoated CNTCOOH (1 mg/mL). Fluorescence emissions were measured at 525 nm for 20 min using a microplate reader Synergy 4 (BioTek). 2.17. Platelet membrane integrity measurement by lactate dehydrogenase (LDH) release assay The release of LDH by PLT was measured using the LDH Cytotoxicity Detection Kit (Takara Bio, Japan) in accordance with the manufacturer’s instructions. PLT were exposed to CNTCOOH, as described above (Section 2.11); at the end of the incubation, all samples were centrifuged at 20,000 g for 15 min. Next, 100 mL of the supernatants (in triplicate) were transferred into 96-well plates and incubated with 100 mL of reaction solution for 30 min, protected from light. Absorbance was measured at 490 nm using a microplate reader Synergy 4 (BioTek).

3. Results 3.1. Proteome analysis of blood plasma protein binding to CNTCOOH The Mascot algorithm utilized to search the obtained MS/MS spectra against the human data base qualitatively identified 35 proteins bound to CNTCOOH (Supplementary Information, Table SI 1). The number of spectra matched by Mascot to the peptides (spectral count) indicates that FBG, HSA and IgG are among the most abundant plasma proteins forming the CNTCOOH corona. All of the other proteins forming the corona listed in Table SI 1 were physiologically consistent with these identifications and included hemoglobin, proteins involved in blood coagulation and proteins related to the immune system. 3.2. Characterization of common human proteins binding to CNTCOOH The plasma protein corona or coronas formed from individual proteins on CNTCOOH were visualized by TEM (Fig. 1). The proteins are presented as a dark shadow surrounding the CNTCOOH on the TEM images. The binding of the proteins to CNTCOOH was evaluated using the fluorescence quenching technique. The efficiency of fluorescence quenching reveals information about the relative accessibility of the protein chromophore groups to CNT [32]. The emission spectra of HSA, FBG, IgG and H1 in the absence and presence of CNTCOOH are shown in Fig. SI 1. CNTCOOH efficiently quenches the fluorescence of the proteins, as evidenced by a progressive decrease in the emission intensity with increasing CNTCOOH concentrations. The shift of the peak intensity wavelength in the fluorescence spectrum (Imax) in Fig. SI 1 (insets) arises from a change in the protein conformation [32,33] due to protein adsorption onto the CNTCOOH. The red shift of Imax observed for HSA, FBG and IgG is symptomatic of a shift in the dielectric properties or the polarity of the local environment of the emitter species [32]. The red shift also corresponds to the relatively more polar environment experienced by proteins after conformational changes. The binding of H1 to CNTCOOH, conversely, causes a blue shift on Imax. Evidently, the local dielectric environment of the adsorbed protein is more polar compared to that of the protein in solution. The fluorescence quenching efficiency, KSV, can be estimated by fitting the dependence of I0/I on the CNTCOOH concentration (Fig. SI 2), based on its fit with the SterneVolmer equation (2) (see

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Fig. 1. TEM images of bare CNTCOOH and CNTCOOH coated with human plasma proteins, HSA, FBG, IgG and H1. Human plasma protein or individual protein coronas on CNTCOOH were prepared by incubating CNTCOOH with blood plasma or protein solutions (1 mg/mL); unbound proteins were removed by washing with Tyrode’s buffer. To visualize the protein coronas as dark shadow surrounding CNTCOOH, samples were stained with uranyl acetate.

Supplementary Information). In accordance with Table SI 2, KSV follows the pattern of H1 > HSA > IgG > FBG. KSV is a measure of the proximity or accessibility of a protein to CNTCOOH and reflects the stability of the association between the species. 3.3. Molecular visualization and analysis of protein surface properties The interaction between negatively charged CNTCOOH and proteins is expected to be dependent on electrostatic interactions with the carboxylic groups and on pep stacking and hydrophobic interactions with the non-functionalized areas of the nanotubes. Though dynamic rearrangements of the protein structure can occur after binding to a foreign surface, the initial process of protein adsorption depends on the native protein surface properties. PyMOL was used to determine the accessible surface area, hydrophobic surface area, electrostatic charge distribution and number of aromatic aminoacids on the surface of the studied proteins available for binding to the CNTCOOH backbone (Fig. 2, Fig. SI 3 and Table SI 3). Electrostatic interactions are expected to play an important role in the protein’s association with the negatively charged CNTCOOH. The electrostatic potentials of the HSA, IgG, FBG and H1 molecule surfaces, as visualized by PyMOL, are shown in Fig. 2A. Due to its high lysine and arginine content, the H1 surface potential is mainly positive, while the areas of positive potential on HSA, IgG and FBG are surrounded by regions of negative potential.

3.4. Circular dichroism of proteins coronas The effect of CNTCOOH on the conformation of the absorbed protein was assessed using CD spectroscopy (Fig. 3). The alphahelix and beta-strand percentage values of the reference proteins were consistent with the PDB data. As shown in Fig. 3 and by the Imax shift in Fig. SI 1, all of the proteins studied showed small but significant conformational changes due to the association with CNTCOOH. 3.5. Protein corona effect on CNTCOOH dispersion and charge Protein binding may cause nanomaterials to self-assemble into higher-order structures [32], thus potentially affecting the overall bio-reactivity of the nanoparticles. Bright field microscopy was used to show the effect of the protein corona on the agglomeration state of CNTCOOH (Fig. 4A). While the HSA corona kept CNTCOOH well dispersed, the IgG and H1 coronas caused the formation of small agglomerates, and FBG triggered the strong and irreversible agglomeration of CNTCOOH. The ability of FBG to agglomerate CNTCOOH was confirmed by the increase in the light transmission of the CNTCOOH suspension, which was stirred in the presence of FBG (Fig. SI 4); the other tested proteins did not induce CNTCOOH agglomeration. In addition to its effect on dispersion, protein binding may lead to changes in the CNTCOOH surface charges. Zeta potential

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Fig. 2. “In silico” visualization and analysis of the protein surface properties by PyMOL. (A) Electrostatic potential on protein surfaces. The distributions of positively and negatively charged domains were calculated using the Adaptive Poisson-Boltzmann Solver (APBS) in PyMOL [30]; electrostatic potentials were obtained at a 150 mM ionic strength, with a solute dielectric of 2 and a solvent dielectric of 78.5. Red represents negatively charged domains, and blue represents positively charged domains. (B) The presence of aromatic amino acid residues on the surface of FBG, HSA, H1 and IgG molecules. The aromatic amino acids are color coded, as follows: Trp orange, Phe yellow, Tyr red and His green. (C) Calculated data from protein surface analysis by PyMOL.

measurements show that the value of the CNTCOOH negative surface charge is lowered by the binding of all of the studied proteins, with the H1 corona providing a positively charged surface for the CNTCOOH (Fig. 4B).

3.6. Effect of CNTCOOH protein corona on human blood PLT PLT activation was evidenced by shape changes, pseudopodia formation, aggregation, membrane budding and the release of PMP.

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Fig. 3. Effect of CNTCOOH on the protein far-UV CD spectrum. (A) CD spectrum of HSA, FBG, IgG and H1 in the absence of CNTCOOH (solid line) and in the presence of CNTCOOH (60 mg/mL, dotted line and 180 mg/mL, dotted dashed line). CNTCOOH are optically inactive (arrow). (B) Calculated fractions of different secondary structures in % [33]. H[r] ¼ regular alpha-helix; H[d] ¼ distorted alpha-helix; S[r] ¼ regular beta-strand; S[d] ¼ distorted beta-strand; Turn ¼ turns; Unrd ¼ unordered structure.

Fig. 5A shows LSCM images of membrane-labeled PLT after 20 min of exposure to 100 mg/mL of CNTCOOH in Tyrode’s buffer. The morphology of PLT treated with FBG-CNTCOOH is comparable to that of untreated control PLT. TRAP and H1-CNTCOOH showed stronger PLT aggregation than did uncoated CNTCOOH, while IgGCNTCOOH did not cause any PLT aggregation, despite the observed PLT fragments. HSA-CNTCOOH-treated PLT showed no signs of aggregation, although damage to the membrane did occur, as the membrane dye also labeled the intracellular membranes. Similar effects were observed for plasma-CNTCOOH-treated PLT. The observations from LSCM were confirmed by several other techniques. Light transmission platelet aggregometry on the

plasma showed that the IgG, HSA and FBG coronas attenuated the PLT aggregating effect of CNTCOOH. In contrast, H1-CNTCOOH showed a stronger PLT aggregating activity compared to uncoated CNTCOOH and had an even stronger effect than did the positive control of 20 mmol/L TRAP (Fig. 5BeC). The effect of the CNTCOOH protein corona on the release of membrane microparticles positive for CD41a and AnnexinV binding (CD41aþAnVbPMP) was evaluated by flow cytometry (Fig. 6Ae B). Complementary results were obtained by analyzing the PMP positive for the PLT activation marker CD62P (CD41aþCD62PþPMP) (Fig. SI 5AeB). Uncoated CNTCOOH, similar to TRAP, induced a marked release of PMP. H1-CNTCOOH triggered an even higher

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Fig. 4. Effect of the protein corona on CNTCOOH agglomeration and charge. (A) Bright-field microscopy images of protein-coated CNTCOOH. CNTCOOH were incubated with protein solutions and deposited on poly-L-lysine coated glass slides by cytospin. HSA kept the CNTCOOH dispersed, while IgG and H1 coronas triggered the formation of small agglomerates. However, FBG initiated a severe agglomeration of CNTCOOH. (B) Concentrations of proteins bound to CNTCOOH and the z potentials of protein-coated CNTCOOH. Means  SD presented, n  3.

PMP release than did uncoated CNTCOOH. Neither HSA-CNTCOOH nor FBG-CNTCOOH induced a significant PMP release. Interestingly, though IgG-CNTCOOH did not induce PLT aggregation, a marked PMP release was observed, similar to uncoated CNTCOOH. CNT were shown to induce oxidative stress in many cell lines and this is one of the main routes for their toxicity [34]. The effect of the protein corona on CNTCOOH-induced oxidative stress to the PLT was evaluated by ROS measurement (Fig. 6D). Only the FBG corona prevented CNTCOOH-induced oxidative stress, most likely by the sequestration of CNT into large agglomerates, thereby preventing their accessibility to PLT. The HSA corona delayed the interaction of PLT with CNTCOOH, causing damage at the same level as would have arisen with uncoated CNTCOOH after 20 min of exposure. The

effect of IgG or H1 alone at the same concentration, as bound to CNTCOOH, was negligible (data not shown). The effect of the protein coronas on the PLT membrane integrity was evaluated by LDH release, as represented in Fig. 6E. IgG- and H1-coated CNTCOOH caused a marked increase in the LDH release compared to the uncoated CNTCOOH. The release of LDH arose from the PLT membrane damage [35]. The HSA corona, compared to the other protein coronas, caused less of an LDH release, but the release was still higher than that observed in untreated controls. To distinguish the effects of H1 and IgG coronas from those of free H1 and IgG in solution, we determined the amount of protein bound to CNTCOOH, as shown in Fig. 4B. Exposing PLT to the same concentration of proteins but without CNTCOOH resulted in no

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Fig. 5. The effect of the CNTCOOH corona on PLT visualized by confocal microscopy and light transmission aggregometry. (A) PLT were labeled with Oregon GreenÒ 488 DHPE, incubated with protein-coated CNTCOOH or TRAP and visualized by LSCM. TRAP and H1-CNTCOOH caused a strong PLT aggregation, and bare CNTCOOH-induced PLT aggregation. The HSA corona and plasma corona attenuated the PLT aggregation activities of CNTCOOH but did not protect the PLT from CNTCOOH injury, as the DHPE green also labeled the internal membranes. FBG caused the agglomeration of CNTCOOH, thereby diminishing the effect of CNTCOOH on PLT; the IgG corona caused PLT fragmentation. The effect of the protein corona was evident on the CNTCOOH-driven induction of PLT aggregation. (B) Typical PLT aggregation curves induced by TRAP (positive control), CNTCOOH, HSA-CNTCOOH, FBG-CNTCOOH, IgG-CNTCOOH or H1-CNTCOOH. (C) Maximum PLT aggregation responses after incubation with TRAP or differently treated CNTCOOH. Means  SD presented, n ¼ 3; ##P < 0.01 versus CTRL; *P < 0.05 versus CNTCOOH treated PLT.

significant effect on the PLT. The fragmentation of the PLT by IgGCNTCOOH was confirmed using IgG preparations from two different manufacturers (not shown). TEM images of the PLT cross-sections are shown in Fig. 7. The morphologies of untreated PLT and PLT treated with FBG-CNTCOOH were typical of a non-activated state, i.e., the PLT had a smooth surface and intact granules. TRAP, H1 and IgG caused strong PLT morphology changes, pseudopodia formation and the release of PMP. H1-CNTCOOH and IgG-CNTCOOH were strongly associated with PLT aggregates and fragments, respectively; HSA-CNTCOOH and plasma-CNTCOOH also interacted with PLT, but to a lesser extent than CNTCOOH, and are mostly found outside the PLT cytosol. 4. Discussion Carbon nanotubes are sp2-hybridized materials; consequently, they are near-universal sorbents for organic compounds in aqueous

phases. Therefore, biological media favor the non-covalent interactions of CNT with a variety of proteins and other biomolecules [36,37]. It has been shown that the main forces involved in protein binding to non-functionalized CNT are pep stacking and hydrophobic interactions [17,23] because the CNT surface is highly hydrophobic. For the negatively charged CNTCOOH, other forces, besides the pep stacking and hydrophobic interactions, are expected to be involved in protein binding. Studies on protein adsorption are important because, in reality, once the NPs enter the bloodstream, the proteins that will immediately coat the NPs are the smallest and most abundant ones; however, the corona should evolve into a more stable protein corona, called the “hard corona,” that is formed by proteins that bind more strongly to the NPs. This is called the Vroman effect [9]. We used HSA, IgG and FBG as the models in our study for two reasons. First, these proteins were identified as major plasma proteins forming the corona on CNTCOOH. Second, the adsorption

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Fig. 6. Effects of the CNTCOOH corona on PLT activation, LDH release and ROS production. (A) Flow cytometric analysis of the CNTCOOH-induced release of PLT membrane microparticles. (B) The release of the PLT CD41aþAnnexinV binding membrane microparticles (CD41aþAnVbPMP) was evaluated, **P < 0.01, *P < 0.05 versus CTRL. CTRL, negative control; TRAP, positive control. PLT suspension in Tyrode’s buffer was exposed to CNTCOOH at a 100 mg/mL final concentration for 20 min. (C) TEM image of the PMP released from TRAP-treated PLT. (D) The production of reactive oxygen species (ROS) in PLT exposed to protein-coated CNTCOOH. HSA-CNTCOOH delayed the PLT response to CNTCOOH, while the IgG and H1 coronas induced a stronger production of ROS. FBG had a protective effect, most likely due to the sequestration of CNTCOOH into aggregates. (E) The effect of the plasma protein corona on the release of LDH by PLT. All nanomaterials tested were at a 100 mg/mL final concentration. Means  SD presented, n  3; ##P < 0.01 versus CTRL; *P < 0.05 versus CNTCOOH treated PLT.

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Fig. 7. Transmission electron microscopy images of PLT cross-section after treatment with CNTCOOH. PLT were exposed to CNTCOOH or protein-coated CNTCOOH for 20 min at RT under gentle agitation. TRAP was used as a positive control. H1 and IgG coronas on CNTCOOH caused severe change in PLT morphology and PMP release. Arrows indicate CNTCOOH.

of these proteins in particular is important for the physiological response to biomaterials and is related to biomaterial blood compatibility [20,21]. We also included the positively charged H1 in our studies. The histones were not part of the plasma protein corona formed on CNTCOOH because the plasma was from healthy donors; circulating histones are only observed in patients where massive cell death is induced such as in chemotherapy or trauma [38]. However, the capability of positively charged proteins to bind

to CNTCOOH was demonstrated by LC-MS/MS analyses showing that Lysozyme A, with a pI similar to H1, is part of the plasma protein corona of CNTCOOH. Interestingly, FBG was the most abundantly bound plasma protein to CNTCOOH. The same observation was found for poly(acrylic acid)-coated gold nanoparticles [39]. Whether carboxylic groups on the surface of nanomaterials favor binding with FBG remains unclear. Compared to HSA, H1 and IgG, the molar amount of FBG bound to CNTCOOH is much lower.

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This difference may be due to the larger size of the FBG molecules and the tendency of FBG to bind in parallel to CNT [17], thus preventing further protein binding. The TEM visualization of the protein coronas formed on CNTCOOH by individual proteins showed that protein adsorption was not uniform along the CNTCOOH; thicker, dark areas can be observed in certain parts of the CNTCOOH and indicate the formation of protein aggregates, as reported previously [24]. The sp3 carbon atoms, which are located at the defect sites along the walls and at the ends of the CNT, facilitate oxidation during the CNTCOOH preparation. Thus, the functionalization of the CNT is likely to be non-homogeneous and contain areas in which a larger number of carboxylic groups bind more proteins than less functionalized areas [9]. The main forces involved in protein binding to nonfunctionalized CNT are pep stacking and hydrophobic interactions [17] because the CNT surface is highly hydrophobic. However, the binding of proteins to CNTCOOH is driven also by other types of interaction. Electrostatic interactions are the strongest type of non-covalent bond and can be expected to play an important role in the adsorption process to the negatively charged CNTCOOH. H1, due to its high lysine and arginine content, is positively charged, and its adsorption onto CNTCOOH is expected to be mainly of an electrostatic nature. HSA, FBG and IgG have isoelectric points mostly in the acidic and neutral range, depending on glycosylation; therefore, at a physiological pH, the global interaction between negatively charged CNTCOOH is predicted to be repulsive. However, HSA, IgG and FBG have areas of positive potential that complement the negative potential of CNTCOOH. Evidently, the protein configuration for the binding must allow the association of positive areas to interact with COOH groups while minimizing the repulsion from the negative potential areas. For hydrophobic and pep stacking interactions, a close proximity of the protein binding sites to the non-functionalized gaps on the CNTCOOH surface is necessary. Hydrophobic and pep stacking are short-range interactions that occur at distances of 3.3e4.0 Å and 4e5.5 Å, respectively [40]. These interactions are sterically hindered by the carboxylic groups on the CNTCOOH surface. Consequently, small protein molecules are more likely to reach the non-functionalized gaps of the nanotubes and to interact with the carbon surface through pep stacking and hydrophobic interactions while contributing to electrostatic interactions. In fact, the increase in protein surface area leads to a decrease in the molar amount of adsorbed protein. HSA has a smaller surface area than that of IgG, which constitutes an advantage for HSA, which can fit on the nonfunctionalized gaps of CNTCOOH. Aside from its size, a protein’s conformational flexibility impacts its binding to CNT. Previous studies showed that rigid ligands bind less strongly to the CNT than flexible ligands [41,42]. Thus, HSA is known to be more conformationally flexible than IgG [43,44], which allows for its assumption of more strategic positions and increased binding possibilities. FBG, which has the largest surface area and is relatively more rigid than HSA, has the smallest molar amount of bound protein measured. These results indicated that the association of proteins with CNTCOOH depends on their charge, molecule size and possibly the conformational flexibility of the proteins. Other thermodynamic factors that affect the affinity of proteineCNTCOOH binding should likewise influence the proteineNP binding process. For instance, the molecular crowding effects of the binding of proteins on nanomaterials are likely to play a role, but this topic is beyond the scope of this manuscript. It is well known that if the surface’s local environment causes even minor perturbations in a protein’s structure, these perturbations might be sufficient to trigger aggregate formation [45]. Thus, if the presence of CNTCOOH provides a slightly destabilizing

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environment for the protein, the stability of the protein molecule may be compromised causing the formation of the aggregates. The degree of conformational changes observed for H1, HSA, IgG and FBG upon binding to CNTCOOH were less than 10% as previously reported [46,47]. Protein binding may cause nanomaterials to self-assemble into higher-order structures [32], thus potentially affecting the overall bio-reactivity of nanoparticles. We observed that the HSA corona kept the CNTCOOH well dispersed, that the IgG and H1 coronas caused the formation of small agglomerates, and that FBG triggered the strong and irreversible agglomeration of CNTCOOH. Similar effects were observed when FBG was exposed to carboxylic acid functionalized gold NPs [48]. In addition to its effects on dispersion, protein binding caused changes in the CNTCOOH surface charge. CNTCOOH negative surface charges were minimized by HSA, FBG or IgG binding, and the H1 corona provided a positively charged surface for the CNTCOOH. We have previously shown that the CNT in blood plasma activate the PLT by a mechanism that involves the CNT penetration into the PLT, which triggers increases in intracellular calcium due to injuries within the dense tubular system [14,15]. Here, we observed that the activating effect of CNTCOOH was modulated by the protein corona. HSA coating has been repeatedly observed to prevent PLT adhesion to foreigner surfaces and thrombus formation [49,50]. Similarly, HSA is able to minimize the interaction of CNTCOOH with PLT, preventing PLT aggregation, PMP shedding, LDH release and delayed ROS production. In fact, HSA-CNTCOOH was mostly found outside the PLT cytosol. The agglomeration state of CNTCOOH is an important property that can decrease the surface area that comes into contact with the cells and tissues. Indeed, we observed that FBG-CNTCOOH agglomerates did not cause PLT aggregation, PMP shedding, LDH release or ROS production. It has been reported that coating CNT with FBG has a protective effect in HUVECs compared to uncoated CNT, though the agglomeration state of the nanomaterial has not been discussed [17]. The H1 corona provides a positively charged surface for the CNTCOOH, which favors interactions with the negatively charged PLT plasma membrane. The aggregating effect of H1 alone in PLT has also been previously described [22], but PLT exposed to H1 free in solution at the same concentration as that bound to CNTCOOH did not cause PLT activation. Surprisingly, IgG-CNTCOOH induces PLT fragmentation and a marked PMP release. These observations were confirmed by testing the IgG coronas from different manufacturers. The effect of IgG adsorbed on the artificial surfaces on PLT function is poorly understood, and both activating [51,52] and non-activating effects [53] have been reported. However, it is known that PLT membranes have specific receptors for the Fc portion of the IgG molecule. While IgG monomers do not have a measurable effect on PLT, IgG oligomers strongly affect PLT function [54]. We have also tested the effect of IgG free in solution at the same concentration that was bound to CNTCOOH, and no effect on PLT was observed. This result strongly indicates that the adsorbed layer of IgG molecules on CNTCOOH resembled the IgG complexes. The importance of this finding warrants further investigation. 5. Conclusions In summary, our study showed that the driving forces behind protein binding depend on CNT surface properties. As opposed to non-functionalized CNT, the association of proteins to CNTCOOH depends on a protein’s charge, size and structural flexibility rather than the number of hydrophobic and aromatic amino acids that are available for binding onto the CNT backbone. The relative amount of

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protein adsorbed on CNTCOOH was found to be higher for smaller and positively charged proteins. Protein binding onto CNTCOOH was shown to affect the agglomeration state and charge of the CNTCOOH, which influenced the interactions with blood PLT and ultimately modulated the pro-thrombogenic properties of CNTCOOH. Therefore, studies regarding plasma protein adsorption are pivotal to the proper characterization of nanomaterials and to the definition and prediction of the toxicological profiles of nanomaterials that come into contact with blood and vasculature. Funding This project was supported in part by an appointment to the Research Participation Program at the Center for Biologics Evaluation and Research administered by the Oak Ridge Institute for Science and Education through and interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration. In addition, the study was supported in part by the grant LH12014 of the Ministry of Education, Youth and Sports of Czech Republic. KH was supported by project of Charles University in Prague: PRVOUK-P24/LF1/3. The study was supported by FDA CORES. Disclaimer These findings and conclusions have not been formally disseminated by FDA and should not be construed to represent any Agency determination or policy. Opinions expressed in this paper are the authors’ and do not necessarily reflect the policies and views of the National Institute of Standards and Technology (NIST), or affiliated venues. Certain commercial equipment, instruments, or materials are identified in this paper only to specify the experimental procedure adequately. Such identification is not intended to imply recommendation or endorsement by the NIST, nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.04.067. References [1] Foldvari M, Bagonluri M. Carbon nanotubes as functional excipients for nanomedicines: II. Drug delivery and biocompatibility issues. Nanomed Nanotechnol 2008;4:183e200. [2] Hirata E, Menard-Moyon C, Venturelli E, Takita H, Watari F, Bianco A, et al. Carbon nanotubes functionalized with fibroblast growth factor accelerate proliferation of bone marrow-derived stromal cells and bone formation. Nanotechnology 2013;24:435101. [3] Hu H, Ni YC, Montana V, Haddon RC, Parpura V. Chemically functionalized carbon nanotubes as substrates for neuronal growth. Nano Lett 2004;4: 507e11. [4] Iverson NM, Barone PW, Shandell M, Trudel LJ, Sen S, Sen F, et al. In vivo biosensing via tissue-localizable near-infrared-fluorescent single-walled carbon nanotubes. Nat Nanotechnol 2013;8:873e80. [5] Madani SY, Naderi N, Dissanayake O, Tan A, Seifalian AM. A new era of cancer treatment: carbon nanotubes as drug delivery tools. Int J Nanomed 2011;6: 2963e79. [6] De La Zerda A, Zavaleta C, Keren S, Vaithilingam S, Bodapati S, Liu Z, et al. Carbon nanotubes as photoacoustic molecular imaging agents in living mice. Nat Nanotechnol 2008;3:557e62. [7] Parpura V. Instrumentation: carbon nanotubes on the brain. Nat Nanotechnol 2008;3:384e5. [8] Williams DF. On the mechanisms of biocompatibility. Biomaterials 2008;29: 2941e53. [9] Tenzer S, Docter D, Kuharev J, Musyanovych A, Fetz V, Hecht R, et al. Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology. Nat Nanotechnol 2013;8:772eU1000.

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