Structural Changes in Apolipoproteins Bound to Nanoparticles

Oct 6, 2011 - pubs.acs.org/Langmuir. Structural Changes in Apolipoproteins Bound to Nanoparticles. Risto Cukalevski,*. ,†. Martin Lundqvist,. †. C...
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Structural Changes in Apolipoproteins Bound to Nanoparticles Risto Cukalevski,*,† Martin Lundqvist,† Cecilia Oslakovic,‡ Bj€orn Dahlb€ack,‡ Sara Linse,† and Tommy Cedervall† † ‡

Biochemistry and Structural Biology, Lund University, Lund, Sweden  Department of Laboratory Medicine, Division of Clinical Chemistry, Skane University Hospital, Lund University, Malm€o, Sweden

bS Supporting Information ABSTRACT: Nanoparticles are widely used in the pharmaceutical and food industries, but the consequences of exposure to the human body have not been thoroughly investigated. Apolipoprotein A-I (apoAI), the major protein in high-density lipoprotein (HDL), and other lipoproteins are found in the corona around many nanoparticles, but data on protein structural and functional effects are lacking. Here we investigate the structural consequences of the adsorption of apoAI, apolipoprotein B100 (apoB100), and HDL on polystyrene nanoparticles with different surface charges. The results of circular dichroism, fluorescence spectroscopy, and limited proteolysis experiments indicate effects on both secondary and tertiary structures. Plain and negatively charged nanoparticles induce helical structure in apoAI (negative net charge) whereas positively charged nanoparticles reduce the amount of helical structure. Plain and negatively charged particles induce a small blue shift in the tryptophan fluorescence spectrum, which is not noticed with the positively charged particles. Similar results are observed with reconstituted HDL. In apoB100, both secondary and tertiary structures are perturbed by all particles. To investigate the generality of the role of surface charge, parallel experiments were performed using human serum albumin (HSA, negative net charge) and lysozyme (positive net charge). Again, the secondary structure is most affected by nanoparticles carrying an opposite surface charge relative to the protein. Nanoparticles carrying the same net charge as the protein induce only minor structural changes in lysozyme whereas a moderate change is observed for HSA. Thus, surface charge is a critical parameter for predicting structural changes in adsorbed proteins, yet the effect is specific for each protein.

’ INTRODUCTION Many industrial and medical applications are based on nanoparticles, and the use of nanoparticles is expected to grow quickly, leading to rapidly increasing exposure to the environment and human body. It is important to consider biosafety aspects before introducing new nanoparticles. However, knowledge of the factors that govern the fate and effect of nanoparticles released into the environment or administered as drugs is still limited. When entering a biological fluid, nanoparticles will be coated with proteins and other biological molecules that build up a “corona”.1 The composition of the corona around nanoparticles will be determined by the concentration as well as on- and off-rate constants for each protein. The corona is not immediately established but will change over time until equilibrium is reached.2,3 If the particles enter a new environment, then the corona will change until a new equilibrium is reached (Lundqvist et al. ACS Nano 2011, 5 (9), 75037509). The corona will create a new particle surface, and its organization may be important to the fate of the nanoparticles and to biosafety aspects. Proteins binding to nanoparticles undergo conformational changes. This further complicates the structure of the corona. A structural change in the protein may lead to either a loss or a gain of function. For example, trypsin bound to polystyrene nanoparticle lose its proteolytic function, probably because of r 2011 American Chemical Society

extensive conformational changes.4 Polystyrene particles modified with amine groups interfere with the blood clotting by interacting with specific coagulation factors (Oslakovic et al., unpublished data). A structural change in the protein could also lead to the exposure of amino acid residues, which are normally buried in the core.57 When fibrinogen binds to negatively charged poly(acrylic acid)-conjugated gold nanoparticles, the consequence can be the activation of Mac-1 receptors on macrophages.8 Structural changes in model proteins adsorbed to nanoparticles have been reported. For example, the structural consequences of the adsorption of human carbonic anhydrase I (HCAI) to small silica particles (6, 9, and 15 nm) were shown to depend on the curvature of the particles.9 An interaction surface with smaller curvature (more flat surface) imposes larger changes in the secondary structure. Karlsson et al. showed that the pH of the solution influenced human carbonic anhydrase II (HCAII) adsorption to silica particles (15 nm).10 Lacerda et al. studied how common human blood proteins were affected by gold nanoparticles of different sizes using absorbance, circular dichroism (CD), fluorescence spectroscopy, dynamic light scattering (DLS), and transmission Received: August 22, 2011 Revised: October 6, 2011 Published: October 06, 2011 14360

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Langmuir electron microscopy (TEM).11 The CD signal was decreased for all proteins, indicating a loss of structure. Apolipoproteins are found in the corona formed around most spherical nanoparticles studied,1218 including polystyrene particles.19 Apolipoprotein A-I (apoAI) is often present and is in some cases the dominating protein in the corona.2,19 ApoAI is an important structural protein in high-density lipoprotein (HDL) particles and binds to a phospholipid monolayer encircling a core of cholesterol and cholesterol esters. The protein is involved in cholesterol metabolism and is important in reducing the risk of coronary heart diseases. ApoAI participates in cholesterol transport and interacts with lecithin cholesterol acyltransferase (LCAT), ABC transporter, and phospholipid transporting protein (PLTP).20,21 The crystal structure of lipid-free apoAI has been solved,22 but in solution the structure is flexible and more conformations are possible.23 Only low-resolution and indirect biophysical and biochemical data are available for the structure of apoAI bound to phospholipids. There are at least three types of proposed structures that it can form with lipids.21 All three forms may exist as HDL matures from small cholesterol-poor discoidal particles to larger cholesterol-rich spheroidal particles. It is not known to what extent the secondary and tertiary structures or function of apoAI or other apolipoproteins are affected when the proteins are adsorbed to nanoparticles. In this study, we investigate the conformational changes at the secondary and tertiary levels for apoAI, apoB100, and apoAI associated with phospholipids in reconstituted HDL (rHDL) when bound to polystyrene (PS) and modified polystyrene nanoparticles with different surface charges. The study is complemented with negatively charged HSA and positively charged lysozyme. The structural changes are followed by circular dichroism (CD), intrinsic tryptophan fluorescence spectroscopy, and limited proteolysis.

’ EXPERIMENTAL SECTION Synthesis and Characterization of Nanoparticles. Polystyrene nanoparticles (plain and COOH- or NH2-modified) are from Bangs Laboratories Inc. (Indiana, U.S.); they were purchased as 10% stock solutions and dialyzed extensively against several changes of Millipore water before use. The size and zeta potential of the polystyrene and COOH- or NH2-modified polystyrene particles were determined by dynamic light scattering using a Malvern Instruments Zetasizer NanoZS (Oslakovic et al., unpublished data). The particle stock solutions were diluted 100-fold with Millipore water before measurement, which was conducted at 25 °C. The sizes of the particles were 23.6 or 224, 27.8 or 224, and 57.1 or 284 nm for PS, PS-COOH, and PS-NH2, respectively. 50 nm copolymer 50:50 NIPAM/BAM (N-isopropylacrylamide-co-Ntert-butylacrylamide) particles were synthesized as previously described.24 Plasma Proteins. ApoAI was purified from human plasma as previously described.25 This sample was used in all experiments unless otherwise explicitly stated. Plasma-purified apoAI was used for the preparation of rHDL as previously described.25 Briefly, phospholipids (phosphatidylcholine) were dried and resuspended in PBS buffer (10.1 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, 1.8 mM KH2PO4, pH 7.5) with n-octyl-β-D-glucopyranoside. Lipids were mixed with apoAI (30:1 phospholipid/apoAI molar ratio) and dialyzed against PBS. Commercial apoAI (A-0722) was obtained from Sigma-Aldrich and dialyzed against PBS before use. Human serum albumin from Sigma (A3782, fatty acid-free, 99% pure) was purified from dimer and contaminating proteins using gel filtration on a 200  3.4 cm2 Sephadex G50 column in 50 mM ammonium acetate buffer at pH 6.5. Fractions containing HSA monomer were pooled,

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lyophilized, and desalted by gel filtration on a G25 Sephadex superfine column in Millipore water. Before experimental procedures were carried out, all proteins were dissolved/diluted in PBS with 0.1 or 1 mM EDTA and dialyzed extensively against the same buffer. Stock solutions of HSA (10 mg/mL), apoAI (1.3 mg/mL), rHDL-apoAI (0.65 mg/mL), apoB100 (1.5 mg/mL), and chicken lysozyme (5.7 mg/mL) were kept at 4 °C. Limited Proteolysis. ApoAI (6 μg) in PBS with 1 mM EDTA was mixed with 50 nm 50:50 NIPAM/BAM copolymer nanoparticles or 24 nm polystyrene, 28 nm COOH-modified polystyrene nanoparticles, or 57 nm NH2-modified polystyrene nanoparticles, and the final volume was set to 12.5 μL with buffer. Particle/protein mixtures were incubated for 1 h on ice before adding 2.5 μL of chymotrypsin (∼2 mg/mL) diluted 10-, 100-, or 1000-fold, yielding a final enzyme concentration of 33, 3.3, or 0.3 μg/mL, respectively, and the samples were incubated on ice. For each protein, a control sample without any protease was studied in parallel. After 1.25 to 1.45 h, the proteolysis was stopped by adding 5 μL of SDS-loading buffer (62.5 mM Tris-HCL, pH 6.8, 10% glycerol, 2% SDS, 5% mercaptoethanol, 0.05% bromphenol blue). The samples were heated to 95 °C, and the proteins were separated via SDS-PAGE on a 15% gel. Protein bands were visualized using Coomassie blue. Selective Binding of Commercial apoAI Species. Commercial apoAI (10 μg) was mixed with 1 mg of PS (224 nm), PS-COOH (224 nm), or PS-NH (284 nm) in a total volume of 20 μL of PBS and incubated for 1 h at 23 °C. Bound protein was separated from unbound protein by pelleting the protein/particle complexes by centrifugation at 13 000 rpm for 5 min. The supernatant was saved for further analysis, and the pellet was washed with 0.5 mL PBS, dispersed, and centrifuged again. The supernatant was discarded, and bound proteins were solubilized by adding 10 μL of SDS-loading buffer and separated with SDS-PAGE on a 12% gel. Protein bands were visualized using Coomassie blue.

Circular Dichroism and Intrinsic Tryptophan Fluorescence Spectroscopy. Increasing amounts, 0 to 0.45 mg/mL, of 24 nm polystyrene, 28 nm COOH-modified polystyrene, 57 nm NH2-modified polystyrene particles, or 1 mg/mL of 50 nm 50:50 NIPAM/BAM copolymer particles in PBS were mixed with proteins, 0.05 to 10 mg/ mL, in PBS + 0.1 mM EDTA (Table SI 1). The nanoparticles were added to the protein solution and incubated for 30 ( 5 min before recording spectra. The CD signal was recorded between 260 and 200 nm in a quartz (QS) cuvette with 1 mm path length at 37 °C using a Jasco J-815 CD spectrometer. The scanning rate was 50 nm/min, the digital integration time per data point (DIT) was 8 s, and the data collected was from an average of five accumulations. No difference was seen in the CD signal after incubation for 5 or 60 min after adding the nanoparticles to the proteins. For the experiments with HSA and oleic acid, 6 mol equiv of oleic acid was allowed to bind to HSA before nanoparticles were added and incubated for 30 ( 5 min. The fluorescence emission spectrum was recorded at 37 °C in 10 or 3 mm QS cuvettes using a Perkin-Elmer LS 50 B luminescence spectrometer between 310 and 420 nm after excitation at 290 nm at a scan rate of 50 nm/min. The excitation and emission slits were set to 3 nm. The data collected was from an average of three accumulations, and the background signal from the nanoparticles was subtracted. The samples were handled in the same way as for the CD spectroscopy. Identical nanoparticle titrations into protein-free buffer, PBS + 0.1 mM EDTA, were made, and the CD and fluorescence signals were recorded. In the case of fluorescence, these data were used for baseline subtraction from the proteinnanoparticle data. Typically, the CD signal-to-noise ratio was higher in the presence than in the absence of proteins. This is especially true for PS-NH2. Therefore, the CD results are reported without subtracting the background signals. Binding Parameters. The data from the fluorescence experiments were used to obtain quantitative estimates of the equilibrium 14361

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Figure 1. Far-UV CD(upper panel) and fluorescence emission spectra (lower panel) of apoAI alone (green) and with different concentrations of (left) PS, (middle) PS-COOH, and (right) PS-NH2. The concentration of nanoparticles is 0.1 (blue), 0.3 (red), or 0.4 mg/mL (black), and the concentration of apoAI is 0.1 mg/mL. dissociation constant, Kd, and the stoichiometry of each protein binding to nanoparticles. The data is formally described by the following equation Ftot ¼ Fp ½p þ Fnp ½np þ Fpnp ½pnp

1 ½np ¼  ðKd þ ½ptot  n½nptot Þ 2 ffi rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 ðKd þ ½ptot  n½nptot Þ2 þ Kd n½nptot þ 4

ð1Þ

where Ftot is the total intensity and Fp, Fnp, and Fpnp are the intensities from free protein, free nanoparticles, and the proteinnanoparticle complex, respectively. [p], [np], and [pnp] are the molar concentrations of the proteins, nanoparticles, and complex, respectively. Assuming that the nanoparticle signal does not change upon protein binding, the intensity from nanoparticles in buffer was subtracted from the total intensity at each titration point, and the following equation was fitted to these data: Ftot ¼ Fp ½p þ Fpnp ½pnp

where [np] is

ð2Þ

We used the following equations ½ptot ¼ ½np þ ½pnp

ð3Þ

n½nptot ¼ ½np þ ½pnp

ð4Þ

Kd ¼

½p½np ½pnp

ð5Þ

Q ¼

½pnp ½ptot

ð6Þ

where n is the number of sites on the nanoparticle, Kd is the dissociation constant, and Q is the proportion of bound protein to total protein concentration (also called the degree of saturation of the protein). Equation 2 can be expressed as ! Fp Kd þ Fpnp ½np ð7Þ Ftot ¼ ½ptot Kd þ ½np

Equation 7 was fitted to the titration data after nanoparticle baseline subtraction.

’ RESULTS AND DISCUSSION Lipoproteins and Apolipoproteins. Apolipoprotein A-I. Far-UV CD spectroscopy (260200 nm) was used to monitor changes in the secondary structure of apoAI in the presence of nanoparticles. In the CD spectrum of apoAI without nanoparticles, there are two minima at 222 and 208 nm (Figure 1). This is indicative of an α-helical structure. Similar spectra have been reported for lipid-free apoAI,26 and in solution, apoAI is thought to have an extended, mostly α-helical structure. Upon binding to PS or PS-COOH particles, the α-helical signal is enhanced, indicating that the structure is stabilized or enhanced (Figure 1). Similar structure enhancement has previously been reported for apoAI bound to phosphatidylcholine (PC) small unilamellar vesicles (SUV)27,28 and to dimyristoyl phosphatidylcholine (DMPC) that resemble HDL.29 The opposite effect is noticed when the apoAI-PSNH2 complex is formed (Figure 1). The CD signal for apoAI decreases, and the trend is more pronounced at a higher concentration of particles. This indicates that the secondary structure of apoAI is disrupted after binding to positively charged particles. The ratio between the signal intensities at the two minima at 222 and 208 nm is increasing when the nanoparticle concentration increases (Table 1 and Figure SI 1). This observation is most pronounced with PS-COOH nanoparticles, for which the ratio increases from 0.91 without particles to 1.07 with 0.3 mg/mL 14362

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particles. Similar changes in the ratio between 222 and 208 have been observed when free α-helices interact to form coiled-coil structures.3033 Fluorescence spectroscopy is used to measure the emission after excitation of the intrinsic aromatic residues. After excitation at 290 nm, most of the emission is derived mainly from the tryptophan side chains. The wavelength maximum and the intensity are influenced by the polarity of the environment around the tryptophan side chains. A shift in the emission spectrum is therefore a sign of changes in the tertiary structure around one or several tryptophan side chains. The emission spectrum of apoAI has a maximum at 340 nm indicating that the protein has a folded conformation (Figure 1). With increasing amount of PS or PS-COOH nanoparticles added to a fixed concentration of apoAI, a small blue shift in the emission spectrum is observed (Figure 1). This indicates that the environment around one or several tryptophan side chains is more hydrophobic after binding to polystyrene nanoparticles. This may be explained by a conformational change in apoAI by which the tryptophan side chains adsorb to or become buried in the hydrophobic polystyrene surface. The intensity at 340 nm versus the particle concentration has the shape of a Table 1. Ratio of CD Intensities at 222 and 208 nma particles

a

ApoAI

rHDL

none

0.91

0.99

PS

0.93

1.02

PS-COOH

1.07

1.10

PS-NH2

1.00

1.02

The concentration of apoAI is 0.1 mg/mL, and the concentration of nanoparticles is 0.3 mg/mL.

binding curve and indicates that binding is saturated at higher concentrations of particles (Figure SI 4). The PS and the PS-COOH nanoparticles have negatively charged surfaces, and to examine the importance of nanoparticle charge, positively charged PS-NH2 particles were added to apoAI. The fluorescence emission spectrum of apoAI together with that of PS-NH2 displays no blue shift compared to apoAI alone, and in contrast to PS and PS-COOH, the intensity increases after adding the particles. ApoAI has been shown to be the major protein in the protein corona around copolymer NIPAM/BAM nanoparticles in human plasma.1 The copolymer particles are thermally sensitive (i.e., they readily disperse at low temperature but aggregate at high temperature). Therefore, the experiments with these particles were performed at 0 or 4 °C. Limited proteolysis can be used to monitor structural changes in proteins. At short reaction times, only the most exposed sites will be cut by the protease. Changes in the protein structure or local or global changes in protein dynamics upon binding to nanoparticles can expose other sites for proteolysis, and a different peptide pattern may be obtained.7 The peptide pattern of apoAI digested by chymotrypsin is shown in Figure 2A. Two protein bands are seen just below the fulllength apoAI band (left enlarged inset in Figure 2A). For apoAI mixed with copolymer particles, only one protein band is seen below full-length apoAI (right inset in Figure 2A), indicating that sites that are exposed in the free protein are protected in the nanoparticle-bound protein. A structural change in the protein could be an explanation of this behavior, or this is observed because the cleavage site is located at the interface with the particle. Interestingly, the same difference is seen between free apoAI and apoAI bound to phospholipids.34 Attempts were made to use limited proteolysis after apoAI was bound to PS,

Figure 2. Limited proteolysis of apoAI with chymotrypsin (A). (Lane 1) Molecular weight standard. (Lanes 29) ApoAI (6 μg) with copolymer particles was digested with increasing amounts of chymotrypsin (lanes 2 and 6, no chymotrypsin; lanes 3 and 7, 0.3 g/mL chymotrypsin; lanes 4 and 8, 3.3 μg/mL chymotrypsin; and lanes 5 and 9, 33 μg/mL chymotrypsin) for 1 h on ice in the (lanes 25) absence or (lanes 69) presence of 50:50 NIPAM/BAM nanoparticles. The arrows in panel A point to enlarged insets. Far-UV CD spectra (B) of apoAI (1 mg/mL) without (O) or with (b) 50:50 NIPAM/BAM (1 mg/mL). Near-UV CD spectra (C) of apoAI (10 mg/mL) without (O) or with (b) 50:50 NIPAM/BAM (10 mg/mL). 14363

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Figure 3. Selective binding of apoAI species by polystyrene nanoparticles with different surface modifications. ApoAI (10 μg) from a commercial source (lane 2) was mixed with 1 mg of PS (224 nm, lanes 3 and 6), 1 mg of PS-COOH (224 nm, lanes 4 and 7), and 1 mg of PSNH2 (284 nm, lanes 5 and 8). (Lanes 35) Proteins bound to particle pellets after centrifugation and washing. (Lanes 68) Supernatant after centrifugation.

PS-COOH, and PS-NH2. No proteolysis was observed in the presence of PS and PS-COOH (not shown). This is probably due to the inactivation of chymotrypsin upon adsorption to the nanoparticle. This has been reported for trypsin on PS particles.4 The structure of apoAI when bound to copolymer nanoparticles was further studied by near- and far-UV CD spectroscopy. The far-UV CD spectrum of apoAI with copolymer particles has only one minimum indicating a change in the secondary structure relative to free ApoAI. The near-UV CD spectrum monitors structural changes around aromatic amino acid side chains. There are only small differences between the near-UV CD spectra of apoAI with or without copolymer nanoparticles (Figure 2), indicating that there are only small changes in the tertiary structure after binding to the particles. Selective Binding of apoAI Species. We have previously observed that apoAI from a commercial source is separated into two or three different species by SDS-PAGE.35 ApoAI with the slowest migration rate corresponds to apoAI purified by standard methods25 and to apoAI purified using copolymer particles35 (Figure SI 3, lane 3). The major band observed in the commercial product must represent another species of apoAI because of either covalent modification or partial breakdown of the protein. Figure 3 shows that polystyrene particles with different surface modifications selectively bind different species of apoAI. PS and PS-COOH apparently bind all populations, but the species with highest migration rate (lower band) appears to have a higher affinity for PS. PS-NH2, however, binds only the species with the lowest migration rate (upper band) even though there is an overwhelming excess of the other apoAI species in the sample. It has previously been shown that the binding of apoAI by copolymer nanoparticles is highly selective.1,2 The proteins bound to copolymer particles in plasma is compared to the total plasma proteins (Figure SI 3, lanes 2 and 3). This selectivity of binding is even more pronounced as the 50:50 NIPAM/BAM copolymer particles selectively bind the apoAI species with the lowest migration rate (upper band, Figure SI 3, lane 5). This and the marked selectivity in apoAI binding to polystyrene particles strongly suggest that there is a highly specific mechanism of the binding of apoAI, and probably of other proteins, to nanoparticles. Small differences in the protein amino acid sequence and/or

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protein modifications and also the material and surface modification of the nanoparticles seem to affect the selectivity. rHDL. ApoAI is the main protein in HDL. It binds to the phospholipids on the surface of the lipoprotein. It has been shown that copolymer particles bind intact HDL particles in human plasma as well as in purified HDL.36 Here we study the structure of apoAI in HDL after binding to polystyrene nanoparticles. We use reconstituted rHDL instead of HDL purified from plasma because HDL in plasma is a very heterogeneous mixture of particles that differ in size as well as protein and lipid composition. In contrast, rHDL is well-defined, which minimizes the risk of recording data resulting from subpopulations. The far-UV CD spectrum of rHDL is similar to that of free apoAI (cf. Figures 1 and 4). There are two minima at 208 and 222 nm indicative of α-helical structure. Adding PS or PSCOOH nanoparticles to rHDL induces only a small increase in the CD signal, indicating that the amount of α-helical structure is similar for free rHDL and when rHDL is bound to nanoparticles. Adding PS-NH2 to rHDL perturbs the native structure (Figure 3). The ratio of the minima at 222 and 208 nm is higher when apoAI is associated with rHDL compared to when it is in the free form (Table 1 and Figure SI 2). These trends have also been observed for apoAI associated with different phospholipids.2729 Adding PS-COOH to rHDL further increases the ratio, which is not the case for PS and PS-NH2. The emission spectrum for rHDL is similar to that for free apoAI. Increasing the amounts of PS and PS-COOH causes a blue shift that is more evident compared to that of free apoAI (Figure 4), but saturation is not complete (Figure SI 5). Also with PS-NH2 the behavior of rHDL is comparable to that of free apoAI. Apolipoprotein B100. Apolipoprotein B100 (apoB100) is a structural protein in lipoproteins other than HDL (i.e., very low density lipoprotein (VLD) and low-density lipoprotein (LDL)) and is found in the corona around polystyrene nanoparticles.19 The far-UV CD spectrum of apoB100 has a single minimum at 223 nm (Figure 5). Others have reported a minimum at 218 nm for apoB100 dissolved in buffers with detergents, indicating a β-sheet structure. For native apoB100 associated with LDL, the minimum is at 216220 nm.37 Walsh et al. studied apoB100 in micelles and vesicles and observed minima ranging from 216 to 228 nm.38 ApoB100 is difficult to dissolve in water-based buffers, and the differences in the spectra can be due to different degrees of aggregation. Adding PS, PS-COOH, and PS-NH2 to apoB100 decreases the CD signal intensity, and the minimum is shifted toward longer wavelengths (Figure 5). This is often seen when proteins aggregate39 and indicates that apoB100 associates with the three kinds of nanoparticles tested and that the amount of β-sheet structure decreases. This is most prominent for PS-NH2 particles. The fluorescence spectroscopy data indicate that the intensity of apoB100 is decreased when PS or PS-COOH is added (Figure 5). When PS particles are added, there is also a blue shift. A slight blue shift can be noticed with PS-NH2 particles together with increased intensity, indicating no structural effects or minor structural effects, and the tryptophan side chains may be protected from solvent quenching through burial at the nanoparticle surface. If instead the protein had been denatured by the positively charged nanoparticle, then a red shift would have been expected. The fluorescence data thus suggest that apoB100 binds to the three polystyrene particles. Apolipoproteins bind to almost all spherical nanoparticles tested and many nanoparticles with other shapes.1218 This suggests that apolipoprotein binding is a general feature and an 14364

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Figure 4. Far-UV CD (upper panel) and fluorescence emission (lower panel) spectra of rHDL alone (green) and with different concentrations of PS (left), PS-COOH (middle), or PS-NH2 (right). The concentration of nanoparticles is 0.1 (blue), 0.3 (red), or 0.4 (black) mg/mL, and the concentration of rHDL is 0.05 mg/mL.

Figure 5. Far-UV CD (upper panel) and fluorescence emission (lower panel) spectra of apoB100 alone (green) and with 0.3 (red) mg/mL PS (left) and PS-COOH (middle) of PS-NH2 (right). The concentration of apoB100 is 0.2 mg/mL for CD and 0.15 mg/mL for fluorescence measurements.

important component in the protein corona. The binding is selective because it occurs in competition with a large range of other proteins in higher abundance, for example, in blood plasma. A structural change in the apolipoproteins after binding to nanoparticles may result in a gain or loss of functions. Apolipoproteins are central to fat metabolism, and there may be important functional implications that will apply to a majority of nanoparticles. We show that apolipoproteins bound to nanoparticles have different structures than free apolipoproteins. The degree of structural change differs between the different apolipoproteins and depends on the surface charge of the nanoparticles. When ApoAI binds to phospholipids to form HDL, there is an increase in the helical structure and the ratio of the intensities at 222 and 208 nm increases. These changes are also seen when

apoAI binds to nanoparticles, indicating that this binding event induces a similar structural change. This is not as pronounced when rHDL binds to the nanoparticles because apoAI bound to lipids already has an increased ratio for the intensities at 222 and 208 nm compared to free apoAI. Furthermore, in the limited proteolysis experiments, there are differences in the peptide pattern between free apoAI and apoAI bound to phospholipids.34 This is also seen when apoAI is bound to NIPAM/BAM copolymer nanoparticles (Figure 2A). The general binding of apolipoproteins to spherical nanoparticles suggests that particle material is not the determining factor governing apolipoprotein binding. Instead, there may be a general structural mechanism of binding to spherical and curved surfaces that govern their binding over other proteins. There are several mechanisms describing 14365

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Figure 6. Far-UV CD (upper panel) and fluorescence emission (lower panel) spectra of HSA alone (green) and with different concentrations of PS (left), PS-COOH (middle), or PS-NH2 (right). The concentration of nanoparticle is 0.025 (purple), 0.1 (blue), or 0.3 (red) mg/mL, and the concentration of HSA is 0.1 mg/mL for CD and 0.2 mg/mL for fluorescence measurements.

how apolipoproteins bind to phospholipids.23,2729,34,40 Moreover, HDL exists in different forms, and the binding might change with the HDL size. Lipoproteins have a diameter of between 10 and 100 nm (i.e., apolipoproteins are evolved to bind spheres in the nanometer size range and may not distinguish between natural and manufactured spheres). HSA. Fatty Acid-Free HSA. HSA is the most abundant protein (35 mg/mL) in plasma. It is a globular protein with mainly α helices and a negative net charge under physiological conditions. HSA is found in small amounts in the corona around PS, PS-COOH, and PS-NH2 particles.19 The interaction of HSA with different nanoparticles has been reported.4146 The far-UV CD spectrum of HSA has two minima at around 208 and 222 nm, indicating the expected α-helical structure. In the presence of PS or PS-COOH nanoparticles, there are no differences or very small differences in the spectra compared to that of free HSA. In the CD spectrum of HSA with PS-NH2, there is a loss of signal, indicating that the helical structure is perturbed (Figure 6). The fluorescence emission spectrum of HSA clearly changes when PS or PS-COOH particles are added (Figure 6). There is a blue shift, and the intensity decreases with increasing particle concentration. The blue shift indicates that tryptophan side chains are buried in a more hydrophobic milieu after binding, probably at the proteinnanoparticle interface. These results are similar to those with apoAI. When HSA is mixed with PS-NH2 particles, the intensity decreases but there is no spectral shift (Figure 6), indicating that HSA does not bind in the same way to PS-NH2 particles. The intensity decrease is often a sign of lower protein concentration in the sample, which can be due to the precipitation of the complex. However, this contradicts the absorbance spectra and fluorescence spectra measured over time (data not shown). The absorbance is higher for the complex than for protein or particles alone, and the fluorescence intensity does not change. If precipitation would have occurred, then the absorbance and intensity should have dropped over time. The intensity at 340 nm versus particle concentration indicates that the binding is saturated at high nanoparticle concentration (Figure SI 6).

Fatty Acid-Loaded HSA. Fatty acids bind to HSA in the bloodstream. Therefore, it is interesting to study fatty acid effects on the nanoparticle binding of HSA. Six molar equivalents of oleic acid was added to HSA before the nanoparticles were added. There is no difference in the CD spectrum when PS and PS-COOH particles are added to oleic acid-bound HSA or to fatty acid-free HSA (Figure SI 9). When PS-NH2 particles are added, the signal for fatty acid-free HSA is reduced, and no effect is seen for HSA with oleic acid. In the fluorescence emission spectra, there is a clear blue shift for HSA with oleic acid compared to fatty acid-free HSA (Figure SI 10). However, in the presence of PS, PS-COOH, or PS-NH2 nanoparticles, the spectra for HSA and HSA with oleic acid are very similar. The spectra with PS-NH2 particles are similar to that for HSA with oleic acid but with a lower intensity. When PS-NH2 particles and oleic acid are mixed, the sample becomes cloudy, probably because the negatively charged oleic acid interacts with the positively charged nanoparticles. Lysozyme. The structural change after binding to negatively or positively charged nanoparticles differs for apoAI, apoB100, rHDL, and HSA. The change is larger when the proteins bind to positively charged particles than to negatively charged particles. The three proteins are negatively charged in PBS (pH 7.5); therefore, it is interesting to compare what will happen if a positively charged protein, lysozyme, is added to the same nanoparticles. Lysozyme is a small (14.3 kDa) protein widely studied concerning its interactions with nanoparticles.4750 The CD signal decreases when PS or PS-COOH is added, indicating that the secondary structure is disturbed when lysozyme binds to these particles, whereas no change in the CD signal is observed when PS-NH2 particles are added (Figure 7). The fluorescence intensity decreases when PS or PS-COOH particles are added. With positively charged particles, no change is observed (Figure 7). This experiment shows that lysozyme undergoes large structural changes when bound to particles of opposite charge. The lack of changes in the fluorescence and CD signal with positively charged particles indicates either that there is no binding or very weak 14366

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Figure 7. Far-UV CD (upper panel) and fluorescence emission (lower panel) spectra of lysozyme alone (green) and with different concentrations of PS (left), PS-COOH (middle), or PS-NH2 (right). The concentration of nanoparticles is 0.1 (blue), 0.3 (red), or 0.4 (black) mg/mL, and the concentration of lysozyme is 0.1 mg/mL.

binding or that lysozyme binds without any major structural changes. There is no simple relationship between the nanoparticle surface charge and the proteins found in their coronas. PS, PSCOOH, and PS-NH2 nanoparticles bind a wide range of plasma proteins, and many proteins, including apoAI, bind to all three particles. However, most proteins under physiological conditions, as in plasma or cells, carry a negative net charge and the nanoparticle charge may influence the bound protein structure. ApoAI is a negatively charged protein that is found in the corona formed around plain PS, negatively charged PS-COOH, and positively charged PS-NH2 nanoparticles in plasma,19 but the effect on the protein structure differs. PS and PS-COOH particles induce helical structure in apoAI whereas PS-NH2 particles unwind the helical structure, suggesting that the structure of bound apoAI depends on the particle surface charge. A consequence is that positively charged particles affect the structure of apoAI more than do negatively charged particles. The effect of charged groups on the particle surfaces was evaluated for two other proteins, HSA and lysozyme, which are globular proteins with different net charge rich in α-helices. Negatively charged HSA binds to all particles, but only positively charged PS-NH2 particles induce differences in the helical structure. In contrast, in the positively charged lysozyme large structural changes are induced only when it binds to particles of opposite charge. Although an opposite charge between particles and protein is not necessary for an interaction, it induces a more extensive change in the protein structure. The majority of proteins and RNA and DNA are negatively charged, but one group of proteins, DNA- and RNA-binding proteins, are often positively charged. Negatively charged nanoparticles entering the cell could interact with positively charged proteins, for example, histones, induce conformational changes, and interfere with the histones' packing and ordering of DNA. There seem to be distinct structural effects at high nanoparticle concentrations. The reason might be the distribution of bound proteins over a larger nanoparticle surface area instead of the protein being “packed” more closely when the nanoparticle

concentration is lower. Structural change in the presence of a large nanoparticle surface area has been reported9,51 (e.g., the shift in wavelength for HSA, as seen here in Figure 6). Another possibility might be that one protein can bind to two nanoparticles, causing a bridging effect. Binding Parameters. The protein corona formed around nanoparticles will be dependent on the on- and off-rates between proteins and particles and on the protein concentrations. Until equilibrium is reached, the corona will change over time. For copolymer particles, the on- and off-rates for the proteins in the corona are known, and corona formation can be modeled.3 HSA with a high concentration but a low affinity dominates the corona in the beginning, but with time, apoAI with a lower plasma concentration but a higher affinity replaces HSA.3 The polystyrene nanoparticles bind a large number of plasma proteins, and a modeling of the corona has not been reported to our knowledge and would need estimates of the rate constants for several proteins. To estimate the affinity and stoichiometry of apoAI, rHDL, and HSA bound to PS or PS-COOH, the proteins were titrated with increasing numbers of particles and the binding event was followed by fluorescence intensity at 340 nm (Figure SI 46). The Langmuir equation (eq 7) was then fitted to the fluorescence data. ApoAI has a higher affinity than HSA and rHDL for both PS and PS-COOH nanoparticles, which agrees well with previous studies of the nanoparticle corona.1,19 (See Figures SI 46 and 8 and Tables SI 2 and 3 for more information about the estimated affinity and stoichiometry and also the theoretical calculated number of proteins bound as a single dense layer to a nanoparticle.) Our results with HSA are comparable to those of others (e.g., for HSA adsorbed to polymer-coated metal nanoparticles (1020 nm),52 gold nanoparticles (20 nm),11 or copolymer particles (70 nm)3,45), and the affinity is in the same range independent of the type of nanoparticle. With the estimated affinity and stoichiometry for apoAI, rHDL, and HSA, the degree of saturation was calculated using eq 6 (Figure SI 7) and was found to approach 1 at the end of most of the titrations, thus our experiments allow us to compare the structure of free and bound protein. 14367

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’ CONCLUSIONS ApoAI is the main structural protein in HDL, which is known as “good” cholesterol transporting excess cholesterol from the tissues to the liver. ApoAI plays an important role in cholesterol transport and metabolism and interacts and activates several other proteins involved. The observed structural changes might disturb the function of the apoAI, which may lead to problems with fat metabolism and higher risks of cardiovascular diseases. Because apoAI binds to many spherical nanoparticles, this may be a general risk in the field of nanoscience. Structural and functional consequences need to be taken into consideration before using nanoparticles in drugs, food, and other commercial products. The structure of apoAI is affected in different ways depending on the surface character of the nanoparticles to which it binds. Charge seems to play a major role in regulating structural perturbations for apoAI, but the same thing is also shown for other plasma proteins. The methods used in this study give not only information about the structural change but also an estimation of the affinity between the different proteins and nanoparticles. ’ ASSOCIATED CONTENT

bS

Supporting Information. Protein concentrations used for experiments with CD and fluorescence spectroscopy. The ratio of ellipticity at minima 222 and 208 nm obtained from the CD measurements. Detailed fluorescence spectra and the Langmuir function fitted to the titration curve. Dissociation constant Kd and the stoichiometry determined from the fitted Langmuir function. Degree of saturation. Theoretical values for number of bound proteins. CD spectra and fluorescence emission spectra of HSA with and without oleic acid bound to nanoparticles. This material is available free of charge via the Internet at http://pubs. acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT This study was supported by the Swedish Research Council, € K (The Research VR, the NanoVaccin Centre (Copenhagen), FLA School in Pharmaceutical Science), the Crafoord Foundation, Lund, and the Marianne and Marcus Wallenberg Foundation. ’ REFERENCES (1) Cedervall, T.; Lynch, I.; Lindman, S.; Berggard, T.; Thulin, E.; Nilsson, H.; Dawson, K. A.; Linse, S. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2050–2055.  (2) Cedervall, T.; Lynch, I.; Foy, M.; Berggard, T.; Donnelly, S. C.; Cagney, G.; Linse, S.; Dawson, K. A. Angew. Chem., Int. Ed. 2007, 46, 5754–5756. (3) Dell’Orco, D.; Lundqvist, M.; Oslakovic, C.; Cedervall, T.; Linse, S. PLoS One 2010, 5, e10949. (4) Koutsopoulos, S.; Patzsch, K.; Bosker, W. T.; Norde, W. Langmuir 2007, 23, 2000–2006. (5) Lynch, I.; Cedervall, T.; Lundqvist, M.; Cabaleiro-Lago, C.; Linse, S.; Dawson, K. A. Adv. Colloid Interface Sci. 2007, 134135, 167–174. (6) Lynch, I.; Dawson, K. A.; Linse, S. Sci. STKE 2006, 2006, pe14. (7) Lundqvist, M.; Andresen, C.; Christensson, S.; Johansson, S.; Karlsson, M.; Broo, K.; Jonsson, B. H. Langmuir 2005, 21, 11903–11906.

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