Large Protein Absorptions from Small Changes on the Surface of

ARTICLE pubs.acs.org/JPCC

Large Protein Absorptions from Small Changes on the Surface of Nanoparticles Morteza Mahmoudi*,†,‡ and Vahid Serpooshan§ †

National Cell Bank, Pasteur Institute of Iran, Tehran, Iran Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran § Department of Cardiology, School of Medicine, Stanford University, Stanford, California 94305-5101, United States ‡

ABSTRACT: Due to their ultrasmall size, nanoparticles possess distinct properties, when compared with the bulk form of the exact same material. These unique properties are now being exploited in biology and biomedicine in order to probe biological systems and deliver biosensors or drugs into the targeted areas. However, relatively little is known about the interaction of nanoscale objects with living systems. In a biological fluid for example, various proteins associate with nanoparticles. The small size and high curvature angles of nanoparticles bias the physicochemical properties, sizes, and amounts of the proteins presented on their surface. The differential display of proteins bound to the surface of nanoparticles can influence the tissue distribution, cellular uptake, and biological effects of nanoparticles. This study is aimed at verifying the influence of surface roughness of nano-objects on the composition of the corresponding protein corona. A novel approach was used to synthesize magnetic coreshell nanoparticles, with smooth or rough gold shells, by creating either uniform or nonuniform polymeric gaps between the core and the shell. The interactions between the SPIONs and human plasma were probed. Transmission electron microscopy, together with atomic force microscopy, confirmed the formation of various surface topographies on the SPIONs. Field cooling magnetization measurements indicated a decrease in magnetization as a result of gold-coating of nanoparticles. Nuclear relaxivity measurements yielded significantly greater transverse relaxivity in SPIONs, indicating the potential application of these nanoparticles as alternative MRI contrast agents, allowing imaging modality. These results, therefore, confirm the significance and potential applications of emerging iron nanoparticles associated with nanoengineered coatings in the field of biomaterials engineering and nanomedicine.

’ INTRODUCTION It is now well-recognized that what cells actually “see”, when interacting with nanoscale objects, is the long-lived “hard” protein corona, which is formed on the surface of nanoparticles dispersed in complex biological media.14 These protein coronas serve as key mediators, signifying the characteristic properties of nanoparticles to the surrounding cells. The composition of such protein layers is stable for longer than the time scale typically required for cells to import nanoparticles; therefore, these polymeric shells possess an important influence in the biological fate of nanoparticles such as their intracellular uptakes and trafficking. Due to their significant role in altering the dynamic composition of the protein corona, physicochemical properties of nanoparticles (e.g., size, shape, surface charge, angle of curvature, porosity, surface crystallinity, heterogeneity, and hydrophobicity/hydrophilicity) have been recognized as key factors, controlling the way that cells or organs see the nanoparticles.57 Among various physicochemical properties, the surface morphology of nano-objects has been shown to profoundly influence biological responses (e.g., activation r 2011 American Chemical Society

of cellular signaling pathways), due to the fact that a slight variation in particles’ surface properties can cause significant changes in the composition of the forming protein corona.812 Thus, quantitative characterization of the influence of nanoparticle’s surface roughness in the amount, structure, conformation, and gradient of adsorbed proteins is necessary for a deep understanding of their corresponding cellular responses (e.g., cellular trafficking in the particles and activation of signaling pathways).1315 The influence of surface topography in the absorption of various proteins onto the films have been investigated by several groups, confirming the important role of surface roughness.1618 However, the influence of this physiochemical property in absorption properties of nanoparticles has not been fully investigated, due to the hardship of the creation of nanoshells with either controllable uniformity or nonuniformity of their outer surfaces.3 Received: June 15, 2011 Revised: August 3, 2011 Published: August 24, 2011 18275

dx.doi.org/10.1021/jp2056255 | J. Phys. Chem. C 2011, 115, 18275–18283

The Journal of Physical Chemistry C

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Figure 1. Schematic representation of the magnetic separation technique, along with the rinsing steps that nanoparticulate solution undergoes in order to obtain the selected nanoparticles.

Figure 2. Schematic representation of the key steps involved in the synthesis of (a) smooth and (b) jagged gold-coated SPIONs with a polymeric gap. Transmission electron microscopy images confirm the formation of smooth (top right) and jagged (bottom right) gold-coated SPIONs with narrow size distribution.

Herein, we present human protein adsorption onto identical nanoparticles with different surface properties (i.e., surface roughness); a new methodology is applied to synthesize magnetic coreshell nanoparticles, with smooth and rough structures, by creating either uniform or nonuniform polymeric gaps between the core (i.e., superparamagnetic iron oxide nanoparticle, SPION) and the shell (i.e., gold coating). Using this approach, and by employing the principles of physical chemistry, the composition of

the protein corona on both smooth and rough surfaces is correlated with the structural data of the protein complexes that are free from the excess plasma. Once isolated from the plasma, nanoparticleprotein complexes are characterized using dynamic light scattering, zeta-potential, one-dimensional (1D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and electrospray liquid chromatography mass spectrometry. We find that the modulation of surface properties of nanoparticles can 18276

dx.doi.org/10.1021/jp2056255 |J. Phys. Chem. C 2011, 115, 18275–18283


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Table 1. Physicochemical Properties of the Manufactured Iron Nanoparticles Particlesa NPs

DH (nm)b

PDIc

ÆDHæ (nm)d

zeta potential (mV)

bare

13.7 ( 2.1

0.29

18.3 ( 3.2

+43.7 ( 1.7

smooth gold-coated SPIONs

27.8 ( 2.6

0.19

34.2 ( 2.2

36.5 ( 1.56

jagged gold-coated SPIONs

28.1 ( 2.8

0.17

37.4 ( 2.7

37.6 ( 1.29 108 ( 3.54

a

Size and zeta potential were measured using dynamic light scattering and particulate microelectrophoresis, respectively. Size measurements are presented as mean ( SD of four samples. b z-average hydrodynamic diameter extracted by cumulant analysis of the data. c Polydispersity index (it is notable that the PDI amounts for the nonaggregated nanoparticles should be lower than 0.3). d Average hydrodynamic diameter determined from CONTIN size distribution.

Figure 3. AFM micrographs of (a) smooth and (b) jagged gold-shell SPIONs. Panels c and d show the image profiles of selected NPs in various spherical directions (upper inset panels), demonstrating smooth and jagged morphologies of the nanoparticles; lower inset panels show the corresponding TEM image of the nanoparticles with different surface shapes (the TEM scale bare is 15 nm).

significantly influence protein corona composition which will require further consideration in the future.

’ MATERIALS AND METHODS Materials. FeCl2 3 4H2O, FeCl3 3 6H2O, diethylene glycol, sodium hydroxide (NaOH), NH2 OH 3 HCL, gold salt (HAuCl4), poly-L-histidine (PLH), and poly(2-vinylpyridine) were purchased from Sigma-Aldrich. PLH and poly(2-vinylpyridine) were used as templates to direct gold nucleation and growth. Phospholipid polyethylene glycol terminated with carboxylic acid (PL-PEGCOOH) was purchased from Avanti polar lipids. Pyridine was obtained from Sinopharm Chemical Reagent Company. Synthesis of the Smooth Gold-Coated SPIONs. In order to obtain nanoparticles with a narrow size distribution, polyol route was employed. Smooth gold-shell SPIONs were produced according to a previous report.19 Briefly, the prepared SPIONs were mixed with PL-PEG-COOH (1:1.5 w/w) in chloroform and remained still until the solvent gradually evaporated. The residual coated SPIONs were heated to 80 C for 5 min and were redispersed in deionized (DI) water via sonication. The obtained materials were collected using a strong magnet and washed

several times with DI water. PLH was added to the solution of SPIONs, and the pH was adjusted to 56, using 0.1 N HCl. After incubation for 60 min, the magnetic nanoparticles (NPs) were collected with a magnet and washed several times with DI water. The obtained solution was mixed with HAuCl4 (1% w/w) for 20 min, with the pH adjusted to 910 using NaOH. Subsequently, NH2OH 3 HCL was added to the solution and mixed well until the color of the colloidal suspension turned to dark blue. The color change was visible in a few minutes. The obtained solution was washed several times, redispersed in DI water using the sonicator, and stored at 28 C. Synthesis of the Jagged Gold-Coated SPIONs. In order to create jagged gold-coated SPIONs, prepared SPIONs were mixed with poly(2-vinylpyridine) and PL-PEG-COOH (1:1.5 w/w; the same concentration of polymers were employed). The preparation procedure was similar to that for the smooth gold shell, except for the final stage, i.e., addition and reduction of gold salts. After PLH addition, the solution containing poly(2vinylpyridine) and PL-PEG-COOH coated SPIONs was mixed with HAuCl4 (w/w 1%) for 20 min, with the pH adjusted to 45 using NaOH. In this pH, poly(2-vinylpyridine), which is a pHsensitive polymer, possesses a folded conformation, whereas the 18277



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PL-PEG-COOH molecule has a stranded shape.20 By reduction of gold using NH2OH 3 HCL, the jagged gold-coated SPIONs were obtained. The resulting solution was washed several times, redispersed in DI water using a sonicator, and kept at 28 C. Interactions of Both Smooth and Jagged Gold-Shell SPIONs with Human Plasma. The interactions of both smooth

and jagged gold-shell SPIONs with human plasma (HP) in the “hard corona” state were probed. It is notable that the human plasma was obtained from 8 volunteers, following the HUPO BBB guidelines,21 at Pasteur Institute in Tehran, Iran. Typically, 100 μL of nanoparticles (concentration of 100 μg/mL) was mixed with 900 μL of HP, followed by incubation at 37 C for 1 h. Since the previous reports had confirmed the formation of the protein corona in a relatively stable manner over a period of 1 h,22 a 1-h time course was selected in this study for evaluation of protein coronas in the samples. In order to probe the composition of “hard corona”, a magnetic separation method was employed. Protein nanoparticle solutions were run through a strong magnetic field using a magnetic-activated cell sorting (MACS) system (see Figure 1). In this case, both smooth and jagged gold-shell SPIONs were fixed inside the magnetic column and the flow-through fractions were removed, respectively. In order to remove the excess (unbound or loosely bound) proteins, fixed NPs were washed three times by PBS. Finally, the column was removed from the magnetic field and magnetic nanoparticles were fully removed and transferred to a low protein binding Eppendorf tube followed by centrifugation at 4 C for 15 min at 2000g. The supernatant was removed, and the samples were stored at 20 C. Characterization Methods. The biophysiochemical properties of both smooth and jagged gold-shell SPIONs were characterized. The morphologies of the magnetic nanoparticles were analyzed by a transmission electron microscope (TEM) operating at 200 kV. To prepare samples for TEM, a drop of the suspension was placed on a copper grid and dried. High-resolution surface images were performed using atomic force microscopy (AFM) to probe the surface morphology and particle size distribution. Samples were imaged with the aid of a Dualscope/ Rasterscope (C26, DME, Denmark), using a DS 95-50-E scanner with a vertical z-axis resolution of 0.1 nm. Dynamic light scattering (DLS) measurements were performed with a Malvern PCS-4700 instrument equipped with a 256-channel correlator. The 488.0 nm line of a Coherent Innova70 Ar ion laser was used as the incident beam. The laser power used was 250 mW. The scattering angles (θ) ranged between 40 and 140. The temperature was maintained at 25 C using an external circulator. Zeta potential determination was performed using a Malvern Zetasizer 3000HSa. Each measurement was an average of six replicates, 1 min each, and repeated five times. Data analysis was performed according to the standard procedures and interpreted through a cumulant expansion of the field autocorrelation function to the second order. Moreover, in order to obtain a distribution of decay rates, a constrained regularization method (CONTIN) was used to invert the experimental data. Magnetization measurements were performed on samples using a superconducting quantum interference device (SQUID) MPMS-XL7 magnetometer. The temperature dependence of the magnetization was studied at temperatures ranging from 5 to 300 K via zero-field-cooling (ZFC) and field-cooling (FC) curves

Figure 4. (a) ZFC/FC magnetization curves for smooth and jagged gold samples; (b) magnetization as a function of applied magnetic field, i.e., hysteresis, at low temperature (T = 5 K). The inset shows a zoom of the curves.

Table 2. Blocking Temperature (TB), Coercive Field (Hc), and Remanence Magnetization (Mr) Obtained from Magnetization Experiments, Together with the Longitudinal (r1) and Transverse (r2) Relaxivities r1 (mM s)1 NPs coating

TB (K)

r2 (mM s)1

HC (mT)

Mr (emu/gFe)

20 MHz

60 MHz

20 MHz

60 MHz

smooth-shaped gold

137.45

16.64

1.3

21.67

10.09

192.4

201.5

jagged-shaped gold

115.54

20.0

3.53

22.35

10.76

193.7

202.3

bare SPIONs

148.54

53.8

5.31

3.41

1.98

18278

42.72

40.96



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Table 3. DLS and Zeta-Potential Data for Smooth and Jagged Gold-Shell SPIONs solutionf sampleV smooth gold-coated SPIONs

Jagged gold-coated SPIONs

PBS

FBS (hard corona)

DH (nm)a

PDIb

ÆDHæ (nm)c

Z-potential

DH (nm)a

PDIb

ÆDHæ (nm)c

Z-potential

31.2 ( 0.3

0.123

37.3 ( 0.9

3.2 ( 0.6

33.4 ( 1.1

0.112

36.8.1 ( 1.4

1.2 ( 0.5

32.1 ( 0.4

0.138

37.8 ( 1.0

7.1 ( 0.9

33.3 ( 0.8

0.193

39.3 ( 2.1

17.9 ( 1.1

6.8 ( 0.7 12.3 ( 1.4

a z-average hydrodynamic diameter extracted by cumulant analysis of the data. b Polydispersity index. c Average hydrodynamic diameter determined from CONTIN size distribution.

isotherms for both smooth and jagged gold-shell SPIONs can be followed. Moreover, the nature and the amount of the most relevant proteins as a function of the surface roughness can be determined. The results would be used in quantitative demonstration of the degree to which the biomolecule corona can change, depending on the nanoparticle’s surface topography. For 1D SDS-PAGE, the stored magnetic nanoparticles were resuspended in 40 μL of fresh PBS followed by the addition of 20 μL of loading buffer, containing 10% DTT. For the LC-MS/MS, the gels were cut on bands and rows, followed by protein digestion into smaller peptides under neutral pH conditions at 37 C using trypsin.

Figure 5. 1D SDS-PAGE hard corona protein profile for smooth and roughgold-coated SPIONs showing the significant effect of surface roughness on protein absorption.

collected at an applied field of H = 50 Oe. The 1H nuclear magnetic resonance dispersion (NMR-D) profiles were determined at room temperature by measuring the longitudinal T1 and transverse T2 nuclear relaxation times, in the frequencies of 20 and 60 MHz for both T1 and T2. In order to define a semiquantitative determination of the compositions of the obtained hard coronas, one-dimensional sodium dodecyl sulfatepolyacrylamide gel electrophoresis (1D SDS-PAGE) and electrospray liquid chromatography mass spectrometry (LC MS/MS) were employed. By looking at the results of 1D SDS-PAGE and LC MS/MS assays, the total binding

’ RESULTS AND DISCUSSION A novel method was used to prepare smooth and jagged goldshell SPIONs (see Figure 2).23 TEM and AFM were used to probe morphology and shape of the prepared SPIONs. The physicochemical properties of the obtained particles are presented in Figure 2 and Table 1. TEM images revealed the smooth and jagged topography of the gold shells in the SPIONs (Figure 2). AFM images of smooth and jagged gold-coated SPIONs are shown in Figure 3. The insets are the corresponding magnified TEM micrographs. The image profiles of the selected particles in various axes show the formation of smooth and jagged gold-coated SPIONs (Figure 3, panels b and d, respectively). Figure 3d together with its TEM inset revealed the formation of a jagged gold ring on the surface of SPIONs with a polymeric gap. The results of zero field cooling (ZFC) and field cooling (FC) magnetization measurements are shown in Figure 4a. To investigate the magnetization behavior as a function of the applied magnetic field, hysteresis experiments in the range of 5T e H e +5T at T = 5K were performed (Figure 4b). All of the measurements were corrected via applying the diamagnetic contributions of sample holder. Table 2 presents the parameters obtained from ZFC/FC and hysteresis curves. As Figure 4, panels a and b, shows, magnetization in the gold-coated samples was lower than that in the bare NPs, and it was higher in the jagged particles when compared with the smooth ones. Figure 4a indicates that the blocking temperature TB, corresponding to the maximum in the ZFC curves, decreased by applying the coatings to the bare SPIONs, whereas the jagged samples showed the highest TB value. Below the TB, the spins froze and the system entered the blocked regime with typical out-of-equilibrium behavior. Hysteresis loops (Figure 4b) indicated small coercive fields Hc (listed in Table 2), together with a residual magnetization Mr in both smooth and jagged NPs, in contrast to the bare SPIONs. The nuclear transverse and longitudinal relaxivities were measured at frequencies of 20 and 60 MHz, at the physiological temperature (T = 37 C). The relaxivity measures the increase of 18279



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Table 4. Representative Hard Corona Proteins Associated with Smooth and Jagged Gold Surfaces in the Plasma Solutions, Measured by LC MS/MSa NSpC

a

gel band Mw (kDa)

uniprot accession number

309

P04275

280

P21333

279

protein identity

smooth

jagged

von willebrand factor

0.08

0.36

alpha-filamin (filamin-1)

1.05

0.83

P02549

spectrin alpha chain; erythrocyte

0.41

0.02

251

P12259

coagulation factor V precursor

1.43

1.64

246

P11277

spectrin beta chain; erythrocyte

0.10

0.07

226

P35579

myosin-9 (myosin heavy chain 9)

4.04

0.05

194

P35579

complement C4-a

3.51

0.13

187

P01024

complement C3

3.78

0.77

130

P0799

thrombospondin-1

2.47

7.31

123

P18206

vinculin (metavinculin).

2.19

1.08

113

P08514

integrin alpha-IIb

3.87

1.73

103

P12814

alpha-actinin-1 (alpha-actinin cytoskeletal isoform)

0.87

0.43

101 90

P02730 P04196

band 3 anion transport protein histidine-rich glycoprotein

0.63 0.71

0.84 7.97

88

P05556

integrin beta-1

0.29

0.14

87

P05106

integrin beta-3

0.74

0.87

85

P0639

gelsolin (actin-depolymerizing factor)

0.30

0.59

82

P16284

platelet endothelial cell adhesion molecule

0.62

0.31

77

P02787

serotransferrin

0.50

0.24

72

P0276

serum albumin

1.60

5.11

72 72

P0267 P01042

fibrinogen alpha chain kininogen-1

1.43 1.25

4.75 0.61

70

P03951

coagulation factor XI

2.20

3.17

69

P07359

platelet glycoprotein ib alpha chain

0.37

1.92

67

P0400

c4b-binding protein alpha chain

0.57

1.13

60

P0267

fibrinogen beta chain

27.89

14.16

53

P16671

platelet glycoprotein 4

0.24

0.35

52

P1090

clusterin

0.49

4.14

49 45

P01871 P05154

ig mu chain C region plasma serine protease inhibitor

5.78 2.00

1.42 3.24

38

P01876

ig alpha-1 chain C region

1.35

0.66

38

P02749

beta-2-glycoprotein

1.35

0.66

36

P04406

glyceraldehyde-3-phosphate dehydrogenase

1.07

0.52

36

P01857

ig gamma-1 chain C region

3.21

1.58

36

P01859


1.78

0.88

36

P01860


2.14

1.05

31

P27105

erythrocyte band 7 integral membrane protein

0.83

2.45

30

P0274

complement c1q subcomponent subunit C

1.71

3.17

30

P02649

apolipoprotein E

1.28

3.59

27

P63104

1433 protein zeta/delta (protein kinase c inhibitor protein 1)

1.90

0.93

27

P02647

apolipoprotein A-I

2.38

5.87

27

P02746

complement c1q subcomponent subunit b

1.90

1.17

23

P23284

peptidyl-prolyl cistrans isomerase b

1.11

0.55

22

P37802

transgelin-2 (sm22-alpha homologue)

0.58

0.57

19

P14770

platelet glycoprotein IX

1.35

3.00

13

P06312

ig kappa chain VIV region

0.99

1.46

12

P01765

ig heavy chain VIII region til

1.07

0.52

11

P01834

ig kappa chain C region

1.17

2.88

11

P01842

ig lambda chain C regions

1.17

2.89

Normalized spectral count (NSpC) values were calculated for each protein hit according to eq 1. 18280



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Figure 6. Normalized spectral counts (NSpC) of (a) the entire proteins within the hard corona composition and (b) proteins of different Mw ranges existing in the hard corona in the smooth and jagged gold-coated SPIONs.

the nuclear relaxation rate per unit of magnetic center (NPs in our case) and verifies the added efficiency by insertion of the magnetic nanoprobe in contrasting MR imaging, in comparison with the natural contrast. Particularly, in superparamagnetic contrast agents, the important parameter is the nuclear transverse relaxivity r2; in this study r2 was measured 201.49 (mM s)1 at ν = 60 MHz and 202.23 (mM s)1 at ν = 60 MHz, for the smooth and jagged gold-coated SPIONs, respectively. Taking into account that the commercial suparparamagnetic contrast agent Endorem at 60 MHz is about 100 (mM s)1 allows us to consider our samples as good MRI contrast agents (see Table 2). In order to probe the protein profile of different nanoparticles, the hard coronas of the samples were evaluated using 1D-gel electrophoresis (see Table 3). As shown in Figure 5, the intensity of the protein bonds in the jagged surface was significantly stronger than that in the smooth surface, indicating the great capability of jagged SPIONs to absorb various biomolecules. This characteristic property can be used as an essential asset in manufacturing nanobioprobes. It is notable that these results were highly reproducible. In addition to the distinct bond

intensities, large differences were interestingly detected when the absorbance of human plasma on the surface of smooth and jagged gold-coated SPIONs was measured (Figure 5). Liquid chromatography mass spectrometry (LC-MS/MS) technique was employed to rationalize the phenomena associated with the effect of surface smoothness on the protein corona composition. The entire 1D SDS-PAGEs bands (Figure 5) were processed and analyzed by MS. In order to obtain the total number of the MS/MS spectra for all of the peptides that are attributed to a matched protein, a semiquantitative assessment of the protein content was conducted through application of spectral counting method (SpC). The normalized SpC amounts of each protein, identified in the MS study of smooth and jagged surfaces, were calculated by applying the following equation:6 0

1

B B ðSpC=Mw Þk C C C 100 NpSpCk ¼ B n @ A ðSpC=Mw Þi

∑ i¼1

18281

ð1Þ


The Journal of Physical Chemistry C where NpSpCk is the normalized percentage of spectral count for protein k, SpC is the spectral count identified, and Mw is the molecular weight (in kDa) of the protein k. Using eq 1, one can expect to obtain the protein size and to evaluate the actual contribution of each protein to the hard corona composition.2 Accordingly, the normalized SpC (i.e., NSpC) values for all of the proteins identified in the hard coronas of both smooth and rough surfaces were determined (listed in Table 4). In addition to the NSpC of each protein, the overall NSpCs of different proteins in a specific molecular weight range (i.e., 7050 kDa) were defined, allowing a better interpretation of the variation of protein profile according to the surface properties of the nano-objects (Figure 5). On the basis of these results, it can be concluded that the surface roughness of the nanoparticles may possess great impact on their corresponding hard corona protein profile. More specifically, it was observed that several proteins in the molecular weight ranges of 310120 and 7030 kDa are preferably adsorbed onto the smooth gold surfaces, whereas proteins in the ranges of 12070 and 3010 kDa are most likely adsorbed onto the jagged gold surfaces (Figure 5b). According to the results presented in Table 4 and Figure 6b, we probed the influence of surface properties on the variation of specific protein composition at the predetermined molecular weight ranges. In comparison with the smooth gold surface, the jagged one showed a significant increase in the NSpC contents of its proteins such as thrombospondin, histidine-rich glycoprotein, serum albumin, fibrinogen alpha chain, and clusterin. Due to the abundance of sharp sectors on the surface, the zeta potential of the jagged gold surfaces was significantly lower and more scattered when compared with that in particles with smooth surface (Table 3). In the case of negatively charged jagged surfaces, with various amounts of charge (i.e., variation of charge in keen edges), although the contribution of entropic gains in protein adsorption may be smaller, the van der Waals interactions, supplemented by electrostatic and H-bonding effects, are usually stronger. Through these phenomena, the amount of several proteins such as myosin-9, complement proteins, thrombospondin, integrins, histidine-rich glycoprotein, serum albumin, fibrinogen alpha chain, clusterin, and platelet glycoprotein IX would significantly change with variation of the surface roughness (Figure 6 and Table 4). The increase in the adsorbed amount of hard corona proteins, with Mw values in the range of 12070, onto the jagged goldshell nanoparticles may be related to the increasing affinity of the most abundant proteins in plasma such as serum albumin and fibrinogen. On the contrary, the affinity of proteins with higher Mw range (i.e., 310120), such as myosin-9 and complement proteins (complement C4-a and C3), is higher for attachment to the surface of smooth gold surfaces rather than the jagged ones. On the other hand, the amounts of apolipoprotein E and apolipoprotein AI are increased in the protein corona of jagged gold surface in comparison to the smooth one. According to the gel and LC MS/MS results, it is revealed that the composition of protein corona is strongly dependent to the surface properties of nanoparticles.

’ CONCLUSION In summary, we demonstrated the significance of the surface morphology of nanoparticles in determination of their protein corona composition. The formation of protein corona is the first stage of a complex series of events that controls multiple

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phenomena occurring at the nanobio interface, such as cellular uptake of nanoparticles and their biodistribution, and cellular responses in vivo. Thereupon, these findings are critical for the design of biocompatible nanoparticles with a high yield in biotherapeutic applications. More specifically, surface decorations of nano-objects are critical in order to provide an in-depth insight into the prediction of biological fate of nanoparticles.

’ AUTHOR INFORMATION Corresponding Author

*Web: www.biospion.com. E-mail: [email protected].

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