Surfactant Titration of NanoparticleâProtein Corona - Analytical

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Surfactant Titration of the Nanoparticle-Protein Corona Daniele Maiolo, Paolo Bergese, Eugene Mahon, Kenneth A. Dawson, and Marco P. Monopoli Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac5027176 • Publication Date (Web): 28 Oct 2014 Downloaded from http://pubs.acs.org on October 29, 2014

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Analytical Chemistry

Surfactant Titration of the Nanoparticle-Protein Corona Daniele Maiolo,a,b,c Paolo Bergese, b Eugene Mahon, a Kenneth A. Dawson a* and Marco P. Monopoli. a* a

Centre for BioNano Interactions, School of Chemistry and Chemical Biology and UCD Conway

Institute for Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland b

Chemistry for Technologies Laboratory, INSTM, Department of Mechanical and Industrial

Engineering, Via Branze and c Experimental Oncology and Immunology Section, Department of Molecular and Translational Medicine, School of Medicine, University of Brescia, viale europa 11, 25123 Brescia, Italy .

Corresponding Authors Marco P. Monopoli ([email protected]) , Tel: 0035317166853 Kenneth A. Dawson ([email protected]), Tel. 0035317162459

KEYWORDS: nanoparticles, protein corona, surfactants, nanobio interface.

ACS Paragon Plus Environment

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ABSTRACT Nanoparticles (NPs) when exposed to biological fluids are coated by specific proteins which form the so called protein corona. While some adsorbing proteins exchange with the surroundings on a short time scale, described as a “dynamic” corona, others with higher affinity and long lived interaction with the NP surface form a “hard” corona, which is believed to mediate NP interaction with cellular machineries. In depth NP protein corona characterization is therefore a necessary step in understanding the relationship between surface layer structure and biological outcomes. In the present work we evaluate the protein composition and stability over time and we systematically challenge the formed complexes with surfactants. Each challenge is characterized through different physicochemical measurements (DLS, zeta potential and DCS) alongside proteomic evaluation in titration type experiments (surfactant titration). 100 nm silicon oxide (Si) and 100 nm carboxylated polystyrene (PS-COOH) NPs cloaked by human plasma HC were titrated with CHAPS (zwitterionic), TritonX-100 (non-ionic), SDS (anionic) and DTAB (cationic) surfactants. Composition and density of HC together with size and ζ-potential of NPHC complexes were tracked at each step after surfactant titration. Results on Si NP-HC complexes showed that SDS removes most of the HC, while DTAB induces NPs agglomeration. Analogous results were obtained for PS NP-HC complexes. Interestingly, CHAPS and TritonX100, thanks to similar surface binding preferences, enable selective extraction of ApolipoproteinAI (ApoAI) from Si NP hard coronas, leaving unaltered the dispersion physicochemical properties. These findings indicate that surfactant titration can enable the study of NP-HC stability through surfactant variation and also selective separation of certain proteins from the HC. This approach thus has an immediate analytical value as well as potential applications in HC engineering.


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INTRODUCTION Dispersed nanoparticles (NPs) can display size distributions in a range comparable to biomolecular building blocks such as proteins, lipids or polysaccharides.1 It has been shown that NPs can specifically engage with cellular membrane receptors2 and given their ability to biologically traffic inside the cell,3 they hold great promise as contrast agents and theranostic vehicles.4 Nanoparticle surface conjugation can allow for the creation of functional nanosystems that can couple biomolecular function with NP physical properties, opening the path to several new applications.5 Additionally, nanoparticles are capable of directly affecting specific cellular mechanisms, such as gene expression6 or apoptosis.7 A crucial challenge to be faced, in order to take these emerging tools to a higher level of development, is to better understand and control the structure of the NP biological interface.8–10 It is now well accepted that a NP (or a NP-biomolecule conjugate) exposed to a biological milieu is covered by a selection of the biomolecular species presenting strong affinity for the NP surface, forming the so called protein corona.11–15 This corona is believed to give a new identity to the NP relevant to biological environment,16–19 modulating biological responses.20,21 For example the protein corona can modulate opsonin mediated NP recognition by macrophages ,3,22 dramatically influencing NP half-life in the bloodstream,14,23–26 and it can also specifically interact with cellular receptors inducing toxic responses.27 Absence or partial coverage of the NP surface by proteins can cause cellular toxicity,16,18,28 while variation in the degree of protein corona “hardening” has been shown to lead to different cellular outcome.29 From the microstructural standpoint, the corona is composed of an external short-lived layer in physicochemical equilibrium with the surrounding environment (the dynamic corona),30 and a long-lived layer directly bound to the NP surface, the hard corona (HC).31–33 The HC is believed to determine the NP identity in the biological environment as it is almost irreversibly bound to


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the NP surface.34,35 Mass Spectrometry studies have revealed that the corona is composed mainly of proteins,13,20,36,37 lipids38 and polysaccharides39 with a relative surface abundance that does not generally reflect their initial concentrations in the biological fluid,40 and the composition is directly linked to nanoparticle surface features such as the curvature13 and chemistry41,42 together with surrounding biological fluid composition.43–45 While recent works have revealed the NP protein corona composition, evolution and physiochemical proprieties, less is known regarding its stability and exchangeability. In this manuscript we describe an analytical approach (Scheme 1) that helps to fill this gap.

Schema 1: Graphical representation of the experimental workflow followed for the Surfactant Titration of the Hard Corona. In particular we applied analytical procedures to study the challenging of the NP-HC through “surfactant titration” of the complexes. This simple and reproducible procedure consists of the systematic exposure of NP-HC complexes to surfactants with different physicochemical properties, followed by their structural characterization (monitoring dispersion, size, change in NP zeta potential) and proteomic approaches (monitoring protein compositional changes).

EXPERIMENTAL Chemicals and Materials: Phosphate Buffered Saline (PBS) is from Sigma Aldrich. 3-[(3Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), Sodium Dodecil Sulphate (SDS), polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether,octyl phenol ethoxylate,


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polyoxyethylene

octyl

phenyl

ether,4-octylphenol

polyethoxylate,Mono

30,TX-100,t-

octylphenoxypolyethoxyethanol,Octoxynol-9 (TritonX-100) and dodecyltrimethylammonium bromide (DTAB) are from Sigma Aldrich. 3-methacryloxypropyltrimethoxysilane (3-MPTS) is from Sigma Aldrich. Nanoparticles: Silica nanoparticles (SiO2 NPs, 100 nm) were synthesized following a standard procedure.46 PS-COOH nanoparticles are from Invitrogen. All nanoparticles were characterized by measuring their size and zeta-potential in physiological buffer before use. NP stock solutions and NPs incubated in plasma were diluted with 10 mM phosphate buffer at pH=7.5 with NaCl 0.15 M and 1mM EDTA (PBS) for all experiments immediately prior to use. Human plasma: Blood was withdrawn from 10-15 different volunteers and collected into 10 ml K2EDTA coated tubes (BD Bioscience). Plasma was prepared following the HUPO BBB SOP guidelines. Briefly, immediately after blood collection, each tube was inverted ten times to ensure mixing of blood with the EDTA, and subsequently centrifuged for ten minutes at 1300 g at 4 ºC. Equal volumes of plasma from each donor were collected into a secondary 50ml falcon tube and then centrifuged at 2400g for 15 minutes at 4 ºC. Supernatant was collected (leaving approximately 10% of the volume in the secondary tube) and it was then aliquoted into 1ml cryovials and stored at -80ºC until use. The whole procedure did not take more than three hours. Following this procedure the plasma protein concentration is estimated to be ~80g/L in agreement with the literature.47 When plasma was used for experiments, it was allowed to thaw at room temperature and centrifuged for 3 min at 16.2kRCF. Thawed plasma was never re-frozen or re-thawed. All data presented are obtained using plasma from one donation session. The blood


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donation procedure was approved by the Human Research Ethics committee at University College Dublin. Sample preparation: Characterization of NP-HC complexes have been performed on samples prepared following previously described procedures. Samples were incubated at

plasma

concentrations (55%), plasma solutions were diluted with PBS keeping the final NP concentration constant and equal to 1 mg/ml. NPs were allowed to incubate with the plasma solutions at 37°C for one hour. To obtain NP-HC complexes, after the incubation in plasma, the samples were centrifuged to pellet the NP protein complexes and separated from the supernatant plasma. The pellet was then resuspended in 500 µl of PBS and centrifuged again for 3 minutes at 18krcf at 15°C to pellet the particle-protein complexes. The standard procedure consists of three washing-steps before resuspension of the final pellet to the desired concentration. This treatment allows us to get rid of the proteins with low affinity for the NP surface (the soft protein corona). Surfactant mediated desorption of the HC: The desorption of the proteins from the HC were obtained incubating the NP-HC complexes in a solution containing increasing concentration of the different surfactant at 37°C for one hour. In particular the following surfactant were used: CHAPS (concentration ranging from 0.01% to 1% w/v), SDS (concentration ranging from 0.01% to 2% w/v), TritonX-100 (concentration ranging from 0.005% to 2% w/v), DTAB (concentration ranging from 0.01% to 2% w/v). The samples were then centrifugated for 30 minutes at 18krcf at 15°C in order to pellet the stripped nanoparticle-protein complexes and resuspended in PBS buffer. SDS-PAGE and MS analysis: Immediately after the last centrifugation step the NP-HC pellet was re-suspended in protein loading buffer [62.5 mM Tris-HCL pH 6.8, 2% (w/v) SDS, 10% glycerol, 0.04M DTT and 0.01% (w/v) bromophenol blue], it was then boiled for 5 minutes at


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100 °C and an equal sample volume was loaded in 10% gel polyacrylamide gel. Gel electrophoresis was performed at 120V, 400mA for about 60 minutes each, until the proteins neared the end of the gel. The gels were stained for one hour in coomassie blue staining [50% methanol, 10% acetic acid, 2.5% (w/v) brilliant blue] and destained overnight in [40% methanol, 10% acetic acid]. After the staining the gel bands of interest have been cut and excised. Then the proteins have been trypsin digested, purified and processed for MS analysis as previously described by Monopoli et al. Gel were scanned using a Biorad GS-800 calibrated densitometer scanner and gel densitometry Dynamic Light Scattering: DLS measurements at θ=173° and zeta potential determination were performed using a Malvern Zetasizer 3000HSa. Each measurement was an average of six repetitions of one minute each and repeated five times. Data analysis has been performed according to standard procedures, and interpreted through a cumulant expansion of the field autocorrelation function to the second order. Differential Centrifugal Sedimentation: DCS measurements have been carried out following method described in literature.32 In the case of the antibody experiment 0.01 mg of anti human ApoAI antibody (Chemicon (Millipore)) has been added to the desidered nanoparticle suspension, incubated for 1 hour at 37 °C and directly injected in the sucrose gradient preequilibrated in the DCS disc. Detailed information about DCS measurements can be found in the Supporting Information. Core-shell model analysis for DCS data: A simple model to analyze data for protein shellcoated particles was developed to get an estimation of the shell thickness. For detailed informations see Monopoli et al JACS.36


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Surface Tension Determination. NP protein corona complexes were dispersed in PBS solution containing increasing doses of the selected surfactant. The measurements of the interfacial tension between air and NP−protein corona complexes were performed through the pendant drop method.48 The experiment were carried out at room temperature (25°C ± 1°) with a CAM 200 tensiometer (KSV Instruments, Finland) equipped with a Navitar camera and employing a cuvette containing water in the bottom to avoid drop evaporation. The images were analyzed using the KSV CAM Optical Contact Angle and Pendent Drop Surface Tension Software 4.04, fitting the drop profile with the circular or the Young– Laplace algorithms. A syringe filled with a solution of NPs or NP−protein complexes connected to a needle was fixed vertically with the needle immersed in air phase. A small amount of solution was injected from the syringe to form a drop. The variation of drop shape with time was captured by automated camera at particular time intervals, and the interfacial tension (γ) was estimated by data fitting using the Laplace−Young equation.

RESULTS & DISCUSSION Surfactant titration: Physicochemical aspects. Plasma protein corona complexes were formed around silica (Si) and polystyrene (PS-COOH) NPs used for this study. The NPs were exposed to human blood plasma at 37°C under agitation,36 and NP-HC have been obtained by centrifugation followed by several washing steps as previously described.11,36 Complexes, isolated following this procedure, were then re-suspended for 1 hour at 37°C in 500µl Phosphate Buffer Saline solution (PBS) or a PBS solution containing increasing concentrations of a given surfactant (surfactant titration). For this study surfactants with different physicochemical features were used, such as CHAPS (a zwitterionic surfactant), TritonX-100 (a non-ionic surfactant),


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SDS (an anionic surfactant commonly used to denature proteins) and DTAB (a cationic surfactant).

Table 1. DLS, ζ-potential and DCS measurements of 100nm pristine NP (silica), Si NPs-HC diluted in PBS (HC) and after exposure to 1% solution of CHAPS and SDS for 1 hour at 37°C (HC CHAPS 1% and HC SDS 1% respectively). Protein corona shell thickness was evaluated by DCS as previously described.36 The errors represent the standard deviation of the mean of three independent replicates.

NP-HC complexes, after re-suspension in PBS and in PBS in the presence of the selected surfactant, were characterized by Dynamic Light Scattering (DLS), zeta potential, and Differential Centrifugal Sedimentation (DCS) as shown in table 1, Figure 1 and Figure S1 for Si. Bare Si NPs displayed a hydrodynamic diameter of 124.9 ± 1.2 nm in PBS, while an increase in the hydrodynamic radius due to the protein corona formation, was observed for Si NP-HC complexes (table 1). The incubation with CHAPS 1% (table 1) or TritonX-100 (Figure S1) induced a slight decrease of the hydrodynamic diameter, while SDS 1% caused a more pronounced decrease of the diameter down to 135.4 ± 0.7 nm suggesting an enhanced protein removal from the Si NP surface. Incubation with DTAB 1% led to particle agglomeration, making the evaluation of the hydrodynamic diameter not feasible. This effect is most likely due to increased surface hydrophobicity with complexation of DTAB on the negatively charged Si NP corona complexes.


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NP protein corona compositional changes were also monitored with evaluation of the NP surface charge by tracking the ζ-potential. While the pristine silica NPs displayed negative charge, plasma protein adsorption to the NP surface resulted in an attenuation of the ζ-potential from −25.1 ± 0.4 mV to − 8.1 ± 0.3 mV. This effect is the fingerprint of protein adsorption and is accurately described elsewhere.31,36,49,50 Treatment with CHAPS 1%, and TritonX-100 1% slightly increased the absolute ζ-potential of the Si NP-HC complexes (Table 1 and Figure S1), while exposure with SDS 1% resulted in a ζ-potential similar to pristine nanoparticles (ζ-potential = −26.6 ± 0.2 mV) suggesting that a more significant removal of the protein coronas takes place. Si NP-HC exposed in 1% DTAB resulted in strong agglomeration making the particle characterization unfeasible. DLS and zeta potential findings suggest that CHAPS and TritonX-100 mildly perturbs the HC protein composition, while SDS is able to efficiently dismantle the HC enhancing the exposition of the pristine NP surface to the solution environment. This was expected, as SDS is a highly effective surface active compound (especially at high temperature) that can desorb proteins from the NP surface by binding and inducing electric charge repulsion between the proteins and the negatively charged surface, even for unfolded proteins.51,52 Strikingly results obtained for Si NP overlap with ones gained for PS-COOH NP where NP plasma exposure drove an increase of the hydrodynamic diameter and of the ζ-potential, whereas NP-HC exposure to “mild” and “strong” surfactants is characterized by a progressive reduction of the corona shell and decrease of the ζpotential (Table S2). NPs size distributions of pristine and NP-HC complexes before and after surfactant exposure were also determined by DCS as it can provides unique details on the NP-HC complex compositional changes related to changes in sedimentation behaviour.53 All the complexes


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persisted as monodispersions and remained stable with the exception of the ones treated with SDS 1% where high exposure of bare surface resulted in partial Si NP agglomeration (Figure 1A). From the DCS size distribution we have evaluated the corona thicknesses where it is possible, as previously shown, to correlate the shift in the apparent size of the NP-HC complexes in relation to the pristine one, with the mass of protein corona adsorbed.36 We found that the initial thickness of the HC was 8.8 nm however it decreased to 4 nm and 1.8 nm after exposure to CHAPS and SDS respectively (Figure 1B), confirming progressive protein corona desorption, in agreement with DLS findings. Protein corona compositional changes have also been evaluated by SDS-PAGE (Figure 1 C-D and Figure S1-3). Interestingly CHAPS and Triton X-100 titrations of the Si NP-HC did not induce significant changes in the corona composition, with the exception of a 30 kDa protein band, whose abundance dramatically decreased after surfactant exposure (Figure 1C-E, Figure S1-2 and table S1). A different scenario occurred when the Si NP-HC complexes were exposed to SDS, where nonspecific and significant protein depletion occurred (Figure 1D-F and Figure S2), with the exception of the triplet of protein bands at 80 kDa, 38 and 35 kDa, that remained associated to a lower extent with the Si NP surface. Therefore, our data suggest that some HC proteins resist this desorption, possibly the ones with highest affinity and directly bound to the NP surface.52,54 Densitometry analysis of the gel lanes confirmed that upon treatment with CHAPS 1% the 30 kDa band decreases by 96%, while the intensity of the other main bands remain substantially unaffected (Figure 1E-F, relative band intensity in Table S1 and Figure S1). Interestingly the rate of the protein desorption after CHAPS exposure to the NP-HC complexes occurred in a dose


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dependent manner, where progressive increase of the surfactant concentration resulted in a more efficient protein release (Figure S2). DTAB exposure of the Si NP-HC complexes resulted in an almost unchanged protein corona (Figure S3) up to the presence of 0.1% of surfactant in solution, however protein of 30 kDa is desorbed at higher concentrations. A different effect was observed for the PS-COOH NP-HC complex surfactant titration. As shown in Figure S4-A CHAPS exposure resulted in a moderate decrease of the NP apparent size (as measured by DCS) compared to the NP-HC complexes, and the decrease in size, thus of the protein corona shell, becomes more significant upon incubation with TritonX100 and SDS. Interestingly the surfactant exposure did not result in a “specific protein corona desorption” as seen for example upon the treatment of Si NP-HC with CHAPS or TritonX-100, but rather in a progressive protein corona removal in accordance with the surfactant physicochemical features.55


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E

F

Figure 1. Effects of detergent exposure on the Si NP-HC complexes. (A) DCS size distributions of the Si NP-HC complexes. The curves refer to pristine 100 nm silica NPs (Silica), NP-HC complexes in PBS (HC), Si NP-HC complexes after exposure to 1% solution of CHAPS or SDS for 1 hour at 37 °C (HC CHAPS 1% and HC SDS 1%, respectively) (B) HC shell thickness evaluated from DCS measurements. (C) Silver staining of a CHAPS Titration SDS-PAGE of the Si NP-HC complexes. Each gel lane identifies the HC in PBS (HC) and HC after incubation with increasing concentrations of CHAPS (from 0.5 % to 1 %). (D) Silver staining of a SDS Titration SDS-PAGE of the Si NP-HC complexes. (E) Densitometry profile of the HC (green line) and after incubation with CHAPS 1% (red line). Densitometries refer to the gel reported in panel C. (F) Band densitometry of HC (green line) and after incubation with SDS 1% (red line). Densitometries refer to the SDS PAGE reported in panel D.


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These findings are supported by other reports in literature where protein corona formed around PS-COOH NPs has been shown to evolve in the presence of competitive protein binders.30,56 In particular gels of corona samples obtained after protein corona desorption with different surfactants report a diverse protein corona pattern/band intensity in agreement with the surfactant surface active properties, thus the higher the surfactant ability to lower the solution surface tension the higher the protein corona desorption (CHAPS

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