Letter pubs.acs.org/NanoLett
Spatial Mapping and Quantification of Soft and Hard Protein Coronas at Silver Nanocubes Teodora Miclăuş,† Vladimir E. Bochenkov,†,‡ Ryosuke Ogaki,† Kenneth A. Howard,† and Duncan S. Sutherland*,† †
Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark Chemistry Department, Lomonosov Moscow State University, Moscow, Russia
‡
S Supporting Information *
ABSTRACT: Protein coronas around silver nanocubes were quantified in serum-containing media using localized surface plasmon resonances. Both soft and hard coronas showed exposure-time and concentration-dependent changes in protein surface density with time-dependent hardening. We observed spatially dependent kinetics of the corona-formation at cube edges/corners versus facets at short incubation times, where the polymer stabilization agent delayed corona hardening. The soft corona contained more protein than the hard corona at all time-points (8-fold difference with 10% serum conditions). KEYWORDS: Silver nanocubes, soft corona, hard corona, localized surface plasmon resonance, protein corona quantification
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has been previously discussed in detail.18 The hard and soft coronas are so classified based on the mean residence time of the proteins in their composition as compared to the duration of the NPs separation steps. The weakly binding, rapidly exchanging proteins that form the soft corona attaching to either the particle surface at short incubation times or to the hard corona at longer incubation times19 are dynamically removed during separation, making them difficult to study ex situ and thus remain a significant experimental challenge. Some in situ studies of individual proteins were able to quantify particle-bound human serum albumin or transferrin through fluorescence correlation spectroscopy without, however, distinguishing between the soft and hard coronas.20,21 Soft protein interactions have been qualitatively investigated in situ, using small-angle X-ray scattering and circular dichroism to show that even weak, brief binding to NPs may affect enzyme structure and activity.22 Recently, a study of the long-lived corona has demonstrated that for some polystyrene and silica spherical nanoparticles hard corona formation is almost instant upon contact with biological fluids, and it is only the ratios between its components that vary with prolonged incubation.23 Other studies support a time-dependent “hardening” of the biomolecule layer, as it is the weakly binding proteins that attach to the nanoparticles first, making it possible to completely wash-off the corona at short incubation times.19 It has been previously shown that preadsorbed coatings around
anoparticles (NPs) have a wide and increasing range of industrial and consumer applications due to specific properties.1,2 Together with the beneficial characteristics, the use of NPs has raised particular concerns in regards to potential harmful effects due to their small size. NPs are both able to penetrate biological barriers3−5 and to interfere with biological processes. During the past decade, extensive studies have been performed in vitro and in vivo in order to better understand the interactions of NPs with biological entities such as tissues,6,7 cells,8,9 and proteins.10,11 It is now accepted that proteins mediate interactions of NPs with cells, by coating particles with a corona that determines the biological identity of the NP and what is “seen” by the cells.12,13 The affinity of different proteins to specific nanoparticles is influenced by NP parameters such as size/curvature,14 shape,15 and surface chemistry.16 Protein interaction with NPs is intrinsically dynamic with exchanges between surface-bound and bulk proteins occurring on a range of time scales. As illustrated in Figure 1A, some proteins exchange slowly, forming the long-lived or hard corona, while others, having lower affinity, are rapidly replaced. The formation of both soft and hard coronas is an equilibrium process, and the protein composition is dictated by the initial concentration of each protein in the incubation medium, as well as its off rate. The long-lived corona has been extensively studied, as it can be removed from the incubation medium together with the particle and analyzed separately.17 This longlived corona is then represented by proteins with mean residence times longer than the duration of the washing steps involved in the separation process. Each washing step involves a decrease of the bulk biomolecule concentration, which in turn shifts the equilibrium between bound and unbound proteins with preferential removal of the faster exchanging fraction, as it © 2014 American Chemical Society
Received: January 23, 2014 Revised: March 3, 2014 Published: March 11, 2014 2086
dx.doi.org/10.1021/nl500277c | Nano Lett. 2014, 14, 2086−2093
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serum (FBS, HyClone) (1−100% FBS for times ranging from 15 min to 24 h). In our experimental setup, we define the hard corona by repeated washing and centrifugation at 15 000g for 15 min, such that all the proteins with mean residence times longer than the duration of the washing step will remain on the NPs as part of the hard corona. This hard corona is analyzed by stripping with sodium dodecyl sulfate and subsequent polyacrylamide gel electrophoresis (see Figures S4 and S5, and section 2 in Supporting Information for detailed description of sample preparation and analysis). The gels showed a characteristic profile of proteins dominated by bovine serum albumin (BSA), as indicated in Supporting Information Figure S4, which is relatively unchanged over time or at different serum concentrations (Supporting Information Figure S5). The overall amount of protein increased over time. In agreement with previous work, the hard corona profile was different from that present in bulk serum, indicating that it does not merely represent the relative bulk protein concentrations, but results from surface concentration of a fraction of the proteins in serum.14,26 Using the bicinconinic acid assay (Thermo Scientific) we quantified the total amount of strongly bound proteins at the surface of AgNCs after 24 h incubation in RPMI-1640 supplemented with 1% FBS, giving 2.24 × 10−15 ± 7.12 × 10−18 μg of protein per square nanometer of particle surface. BSA is the most abundant protein in serum and known to bind to a wide range of NPs, including silver.27 It has been previously shown that BSA attaches to surfaces, forming a monolayer of approximately 7 nm thick,28 which fits with the proposed dimensions of this protein29 as well as with its hydrodynamic diameter.30 Having albumin as a model and a 7 nm monolayer thickness indicating a configuration with a protein footprint of 26.5 nm2 (taken from the protein size of 8.4 × 8.4 × 8.4 × 3.15 nm of the native conformation and an end-down binding position), the amount of protein measured gives a surface coverage of ∼54 ± 1%. This is remarkably close to the coverage of a jammed layer (54.7%) from a random sequential absorbance (RSA) model for monodispersed ellipsoids,31 which has been used to describe protein monolayer limits. In our experimental system, there are multiple protein types and likely multiple orientations at the nanoparticle surface, but the correlation to the RSA jamming limit strongly suggests that the hard corona formed a single, densely packed monolayer with 7 nm as a reasonable estimate for the thickness. The presence of a ∼7 nm thick monolayer of hard corona was further supported by TEM data collected after the same 24 h incubation and washing of unbound and weakly bound proteins. A typical image is shown in Figure 1C, and a rough estimate of the biomolecule layer thickness of about 3.5 nm in the dry, uranyl acetate-stained sample correlates with our model. A decrease of about 50% of the layer thickness is consistent with the collapse and densification of a protein layer upon drying. We have utilized optical resonances intrinsic to the NPs as an in situ sensor to quantify protein binding. Nanoparticles of a number of metals (e.g., Au, Ag, Al, Pt, Pd, Cu) support LSPRs through collective oscillations of electrons. These plasmon resonances have strongly confined optical fields that extend tens of nanometers away from the metal surfaces, as well as optical properties that are dependent on local surrounding refractive index (RI), making them widely applied as sensors.32,33 We make use of the localized nature of the surface plasmon resonances at AgNCs and the sensitivity to the surrounding RI to quantify the amount of protein binding to
Figure 1. (A) Schematic representation of a silver nanocube surrounded by both slowly exchanging and rapidly exchanging proteins in a two-layer model. TEM image of (B) PVP-coated AgNCs and (C) protein-coated AgNCs with an ∼3.5 nm thick hard corona after 24 h incubation (RPMI-1640 with 1% FBS) and PBS washing; the samples were stained with 1% uranyl acetate solution. The arrows point to examples of the organic layer before and after incubation.
metal NPs influence the equilibrium constants for the binding of specific proteins.24 Here, we develop an in situ method to quantify both strongly- and weakly bound proteins at NPs based on localized surface plasmon resonances (LSPRs). We use polyvinylpyrrolidone (PVP)-stabilized silver nanocubes (AgNCs) to show that binding kinetics of proteins can be altered at different locations of the particle surface. Furthermore, we establish the role of the polymeric stabilizing agent on the onset of corona hardening (hard corona formation compared to soft corona). As the experiments were performed in media and at time-scales relevant for in vitro studies, our results provide a step toward further understanding both protein-NP complexes and biomolecule-mediated NP-cell interactions. We synthesized AgNCs using a polyol method described elsewhere.25 The NPs were stabilized by a layer of PVP (SigmaAldrich), which can be seen in the uranyl acetate-stained transmission electron microscopy (TEM) image in Figure 1B. Detailed descriptions of the synthesis and characterization of the particles are provided in the Supporting Information (Section 1.1. and Figure S1 for synthesis; Section 1.2., Figures S2 and S3 for characterization). We characterized the hard (long-lived) protein corona that forms at AgNCs after exposure to cell culture media (RPMI1640, Invitrogen) containing heat-inactivated fetal bovine 2087
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the NPs. By washing and resuspension of the AgNCs in buffer, we isolate particles with the associated hard protein corona, which can be compared to nanocubes devoid of protein corona. The principle for using LSPRs to detect changes in the RI around metal nanoparticles is described in Supporting Information Figure S6. The extinction spectrum of AgNCs exhibits several LSPR peaks,34 two of which are more prominent: a strong, low energy (LE) peak corresponding to a dipole plasmon mode and a weaker, higher energy (HE) peak that results from quadrupole resonance excitation. The position of these signals depends on the size of the cube, but in our system they are localized around 419 and 352 nm respectively, for the particles suspended in Milli-Q water. When incubated in RPMI-1640 supplemented with 1% FBS, the wavelengths of the maximum absorbances red shift, as a consequence of protein binding to the NPs and associated increase in the local RI, as can be seen in Figure 2A. The shift is more pronounced with prolonged incubation times due to an accumulation of protein mass at the nanoparticle. No corresponding peak shifts were observed for AgNCs resuspended in buffer or RPMI without added serum. Upon washing, a minor fraction of the NPs agglomerated, giving a small peak at longer wavelength (570 nm); this agglomeration was also observed in the size data determined by Nanoparticle Tracking Analysis (see Supporting Information Figure S3 and Table S2). In order to better understand the two LSPR signals characteristic for AgNCs, finite-difference time-domain (FDTD) simulations were performed, using the FDTD Solutions software, on a model cube with 35 nm edge length and rounded corners with a curvature radius of 11 nm. The field distributions for the two main plasmon resonances are represented in Figure 2B,C, and the sensitivity of these signals expressed as plasmon decay is presented in Supporting Information Figure S7. As it can be seen, the near field is located mainly at the cube facets for the quadrupole mode and at the edges and corners for the dipole mode. The difference seen in the shape of the HE peak, when comparing experimental and simulated spectra, can be explained by the limitations of the simulation system, where a single orientation of the cubes is employed, whereas manifold orientations exist in the experimental setup. Furthermore, in practice the NPs have a slightly larger size (edge length 37.5 nm) and a certain degree of polydispersity, which may account for the more pronounced HE signal.35 We simulated the optical response of the AgNCs upon protein binding by assuming that a 7 nm thick long-lived protein layer develops over time. Using a protein film density calculated from the experimentally determined surface density of protein (denoted maximum protein coverage) and an average refractive index contribution for the proteins from that of BSA, the simulated RI shift calculated matched well the experimentally observed shift for formation of the hard corona after 24 h of incubation in RPMI + 1% FBS (insets in Figure 2A,D). By simulating 7 nm films of increasing refractive index, we could generate a calibration curve (see inset in Supporting Information Figure S8B), allowing correlation of LPSR peak shifts to changes in RI and protein surface density. The formation of the protein corona is a dynamic process, so we performed studies at a range of time-points from 15 min to 24 h. Briefly, the particle stock suspension was diluted down to a concentration of 10 μg/mL in RPMI with 1% FBS in tissue culture plates. The samples so obtained were kept in an incubator, at 37 °C and 5% CO2 atmosphere, for the desired times. LSPR measurements were carried out on a Shimadzu
Figure 2. (A) Experimental absorbance spectra of AgNCs (10 μg/mL) in water (black) and after incubation (RPMI-1640 + 1% FBS) for 1 h (red), 8 h (light green), and 24 h (blue) with inset showing the shifts of the dipole signal. (B) Energy plot of the near field pertaining to the quadrupole at the surface of a 35 nm edge model AgNC. (C) Energy plot of the near field pertaining to the dipole at the surface of a 35 nm edge length model AgNC. (D) Absorbance spectra for a model AgNC covered by a 7 nm thick layer of proteins with increasing RIs; inset showing the red shift for the dipole signal.
UV−visible-NIR spectrophotometer (UV-3600), working in absorbance mode. Samples were prepared in triplicate and three measurements were performed on each for every timepoint; we show here an average of these measurements. Spectra were collected for the stock particle suspension diluted in phosphate buffered saline (PBS) and for the incubated samples before washing and after removal of unbound and weakly bound proteins by centrifugation and PBS washing. The dipole peak position was determined by deriving the spectral plots using the Savitzky-Golay method (SpecManager software, ACD Labs) and establishing the zero cross point. The position of the signal pertaining to the quadrupole was assessed after fitting of the spectra using the Origin 8 software (OriginLab). The positions of the dipole/quadrupole peaks at different times 2088
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Figure 3. Time evolution of the quadrupole (A) or dipole (B) plasmon signal of AgNCs upon incubation in RPMI-1640 + 1% FBS; blue, before washing of loosely bound proteins; red, after PBS washing of weakly bound proteins; the inset highlights a decrease in the plasmon signals around 60 min incubation and the onset of the hard corona (C) ToF-SIMS signal characteristic of protein (tryptophan fragment), collected for samples after no incubation (blue), 1 min (green), 1 h (orange), and 12 h (red) incubation respectively in RPMI-1640 + 1% FBS. (D) ToF-SIMS signal characteristic of PVP, collected for samples after no incubation (blue), 1 min (green), 1 h (orange), and 12 h (red) incubation, respectively, in RPMI-1640 + 1% FBS. (E) Time evolution of the amount of proteins (expressed as μg per m2 of particle surface) in the hard corona (red) and soft corona at 2 h (dark green, single-layer model) and 24 h (light green, two-layer model) around AgNCs.
before and after washing are shown in Figure 3A,B respectively. As mentioned above, we defined the hard corona as that formed by proteins with a mean residence time longer than the 15 min required for the washing step, while the soft corona is represented by the proteins having shorter residence times and thus being removed during the washing procedure. Furthermore, we acknowledge that during the incubation times ranging from 15 min to 24 h, the attachment of soft corona constituents shifts from binding directly to the particle surface (short incubation) to binding to the constituents of the hard corona layer (long incubation), as this hard corona layer is also being formed during this period. Prior to centrifugation and washing, the particles are covered by both hard and soft coronas. A clear trend is observed for both the LE and the HE signals before and after washing, namely the increase in the wavelength of the peak positions over time, as can be seen in Figure 3A,B. As expected, the slope tends to level-off toward long incubation times, suggesting an equilibrium coverage of the particle surface at 24 h. Longer term exposure experiments showed no significant peak shifts from 24 to 36 h (data not shown). There were some differences in the signals in the first hour; however, after 120 min, the hard corona was clearly accumulating significantly over time. The amount of protein within this hard corona was quantified using a calibration curve (see Supporting Information Figure S8B) generated from the electrodynamic simulations of the optical response of AgNCs. We were able to extract the thin film sensitivity for the dominating dipole peak (LE). The quantification was done taking into account the localized nature of the plasmon resonance and assuming a densification of a 7 nm thick protein film of BSA-like protein and is presented in Figure 3E (expressed as micrograms of protein per square meter of particle surface). The surface density at 24 h is consistent with a
complete dense layer of protein as suggested by the RSA model. The limitations of the FDTD calculations prevent us from quantifying the HE peak shifts explicitly, thus we cannot rule out that the experimentally observed shifts in wavelength of the LE/HE arise from different amounts of protein at different locations on the AgNCs. The amount of protein present within the hard corona increased substantially (by a factor of 8) over a period of 2 to 24 h, without a substantial change in the composition (as judged by the profile of the gel seen in Figure S4 in Supporting Information). This indicates that, at the earlier time-points, the hard corona likely only covered a relatively small proportion (albeit increasing) of the NP surface. The lack of substantial change in the composition of the protein layer during this time period may indicate that the increase of hard corona does not result from addition of different proteins with stronger adhesion at the surface but rather a change of binding strength of the attaching proteins over time. As the hard corona only covers a relatively small proportion of the surface (
