Nano Res

1

Quantitative study of protein coronas on gold nanoparticles with different surface modifications

Menghua Cui, Renxiao Liu, Zhaoyi Deng, Guanglu Ge, Ying Liu (), and Liming Xie () Nano Res., Just Accepted Manuscript • DOI: 10.1007/s12274-013-0400-0

http://www.thenanoresearch.com on December 14 2013

© Tsinghua University Press 2013

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Nano Research

DOI 10.1007/s12274-013-0400-0

1

Quantitative Study of Protein Coronas on Gold

Nanoparticles with Different Surface Modifications

Menghua Cui, Renxiao Liu, Zhaoyi Deng, Guanglu

Ge, Ying Liu* and Liming Xie*

Key Laboratory of Standardization and Measurement for

Nanotechnology of Chinese Academy of Sciences,

National Center for Nanoscience and Technology, Beijing

100190, China

Page Numbers. The font is

ArialMT 16 (automatically

inserted by the publisher)

For bovine serum albumin, transferrin and fibrinogen on gold

nanoparticles (AuNPs), PEG surface modification showed no

protein adsorption. For other surface modifications, the

interaction between protein and AuNPs are strongly dependent

on both surface modification and protein.

Liming Xie, www.nanoctr.cn/xie

2

Quantitative Study of Protein Coronas on Gold Nanoparticles with Different Surface Modifications

Menghua Cui, Renxiao Liu, Zhaoyi Deng, Guanglu Ge, Ying Liu (), Liming Xie () CAS Key Laboratory of Standardization and Measurement for Nanotechnology, National Center for Nanoscience and Technology,

Beijing 100190, P. R. China

Received: day month year / Revised: day month year / Accepted: day month year (automatically inserted by the publisher) © Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2011

 

ABSTRACT   Protein corona provides the biological identity of nanomaterials in vivo. Here we have used dynamic light

scattering (DLS) and transmission electron microscopy (TEM) to investigate the adsorption of serum proteins,

including bovine serum albumin (BSA), transferrin (TRF) and fibrinogen (FIB), on gold nanoparticles (AuNPs)

with different surface modifications [citrate, thioglycolic acid, cysteine, polyethylene glycol (PEG, Mw=2k and

5k)]. AuNPs with PEG (5k) surface modification showed no protein adsorption. AuNPs with non-PEG surface

modifications showed aggregation with FIB. AuNPs with citrate and thioglycolic acid surface modifications

showed 6-8nm thick BSA and TRF coronas (corresponding to monolayer to bilayer proteins), in which the

microscopic dissociation constants of BSA and TRF protein coronas are in the range of 10-8 to 10-6 M.

KEYWORDS Protein corona, gold nanoparticle, dynamic light scattering, transmission electron microscopy, surface

modification

Nanoparticles with size of 1 to 100 nm have

potential applications in bio-imaging[1,2], drug

delivery[3] and cancer therapeutics[4,5]. When

nanoparticles contact with biological fluids, protein

coatings (so-called ‘protein coronas’) are formed on

nanoparticles[6]. Protein corona changes the size of

nanoparticles, alters the surface properties of

nanoparticles and then governs the interactions

between nanoparticles and cells. As a result, protein

corona determines the fate of nanoparticles in

biological system[6]. Therefore, the dynamics and

thermodynamics of protein coronas are of

fundamental importance to various bio-applications

of nanoparticles.

Many studies have been conducted to investigate

interactions between nanoparticles (including gold

Nano Res DOI (automatically inserted by the publisher) Research Article Please choose one

———————————— Address correspondence to Liming Xie, xielm@nanoctr.cn; Ying Liu, liuy1@nanoctr.cn

3

nanoparticles (AuNPs)[7-11], quantum dots

(QDs)[12,13], FePt nanoparticles[12,14,15], polymer

nanoparticles[16,17]) and plasma proteins

(including serum albumin[7,9,10,12,13,17],

transferrin (TRF)[14,16], fibrinogen (FIB)[9,11],

γ-globulin[9] and apolipoprotein[15]). Generally,

protein adsorption on nanoparticles can be

described by the Hill equation[10,12],

where θ is protein coverage on nanoparticle surface;

[Protein] is the free protein concentration; 'DK is

the microscopic dissociation constant (equal to

protein concentration at half coverage); n is Hill

coefficient. For positively/negatively cooperative

absorption, n is larger/less than 1, respectively. For

serum protein adsorption on nanoparticles, 'DK is

usually in the range of 10-8 to 10-3 M and n is close to

1[7,9,10]. For example, 'DK is ~10-6 M and n is ~0.8

for BSA adsorption on citrate-stabilized AuNPs[9].

Dynamically, protein coronas consist of

irreversible, strongly bounded ‘hard coronas’ and

reversible, weakly bounded ‘soft coronas’, which

were evidenced by fluorescence correlation

spectroscopy (FCS) study[16] on TRF adsorption on

sulfonate and carboxyl polystyrene nanoparticles.

Many factors, such as nanoparticle-protein

interactions, protein abundance and even

protein-protein interactions, affect the composition

and structure of protein coronas. Similar to protein

coronas determining the biological identity of

nanoparticles[6], surface properties of nanoparticles,

such as surface chemical groups and surface

electrostatic potential, may govern interactions

between nanoparticles and proteins[18,19], though

the size of nanoparticles also affects the interactions

to some extent[9,18]. The interactions between

proteins and nanoparticles with different surface

modifications are complicated. It is accepted that

polyethylene glycol (PEG) functionalization can

decrease protein adsorption[10,20]. Much work has

focused on carboxyl functionalized nanoparticles,

such as citrate-stabilized AuNPs[9],

carboxyl-polymer-warped FePt nanoparticles[12],

carboxyl-functionalized QDs[12] and carboxyl

polystyrene nanoparticles[16]. Little work has been

done on protein coronas on other surface

modifications. Here we have investigated protein

(BSA, TRF and FIB) coronas on AuNPs with

different surface modifications [citrate (physically

adsorbed), carboxyl, carboxyl plus amine and PEG]

by dynamic light scattering (DLS) and transmission

electron microscopy (TEM). Microscopic

dissociation constant 'DK , Hill coefficient n and the

thickness of protein coronas have been obtained.

As-bought citrate-stabilized AuNPs show a core

diameter distribution of 38.6±3.0 nm (imaged by

TEM, Figure 1a and 1b) and a hydrodynamic

diameter of 43 nm (measured by DLS, Figure 1d).

Slightly larger hydrodynamic diameter measured

by DLS could be due to citrate adsorption and

electrical double layer on the AuNP surfaces.

UV-Vis characterization also showed plasmonic

absorption at ~530 nm (Figure 1e). Ligand

exchange[21] and then dialysis were used to

prepare AuNPs with different surface modifications

(Figure 1c). Thioglycolic acid, cysteine,

methoxypoly(ethylene glycol) thiol

(HS(CH2CH2O)nCH3, Mw=2k, 5k) were used to

prepare AuNP-SCH2COOH (AuNP-COO-, at

pH=7.4), AuNP-SCH2CH(NH3+)COO- (at pH=7.4,

AuNP-Cys), AuNP-S-(CH2CH2O)nCH3 (Mw=2k)

[AuNP-PEG(2k)] and AuNP-S-(CH2CH2O)nCH3

(Mw=5k) [AuNP-PEG(5k)]. Since the binding of

citrate on gold surface is weak, thiols can replace all

citrates on AuNP surfaces via strong Au-S

bond[21,22]. UV-Vis characterization revealed that

neither peak broadening nor new peak at longer

wavelength was observed (Figure 1e), indicating no

aggregation.

Figure 2 (top panels) shows typical DLS correlation

curves for surface modified AuNPs at different BSA

concentrations. In Figure 2e, no hydrodynamic

diameter increase was observed, indicating no BSA

adsorption on AuNP-PEG(5k). In contrast, for

citrate-AuNPs, AuNP-COO-, AuNP-Cys and

AuNP-PEG(2k) (Figure 2a-d top panels), the

correlation curve shifted to longer lag time at higher

BSA concentrations, indicating larger particle

diameters. And the DLS correlation curves at

different BSA concentrations show a similar drop

'

[Protein] (1)

[Protein] ( )

n

n nDK

4

slope as lag time increases, indicating little changes

in diameter distribution (polydispersion index data

in Figure S-1). Cumulative fitting of the correlation

curves can give nanoparticle diameters at different

BSA concentrations (Figure 2 bottom panels). For

citrate-AuNPs, AuNP-COO-, AuNP-Cys and

AuNP-PEG(2k), the diameter increased deeply at

BSA concentration of ~50 nM. The total diameter

increase was ~16 nm, corresponding to a protein

corona thickness of ~8 nm. Considering the

hydrodynamic size of BSA[23] is 14×4×4 nm3, an

8nm-thick corona might correspond to a ‘titled’

conformation of BSA on AuNP surface or two ‘flat’

layers of BSA[10,12].

To quantify interactions between BSA and AuNPs

with different surface modifications, a model[12],

was adopted, where dz([Protein]) and dz(0) are

hydrodynamic diameters of AuNPs with/without

protein, respectively, c is a scaling constant. Since

bounded protein molecules should be much less

than free protein molecules, total protein

concentration was used as free protein

concentration. Fitting hydrodynamic diameter

dependence on protein concentration gives 'DK

and n values for protein-nanoparticle interactions

(Figure 2 bottom panels). Citrate-AuNPs shows a

lager 'DK and an n value of less than 1.

AuNP-COO- and AuNP-Cys show a smaller 'DK

and an n value of larger than 1, indicating a

stronger and positively cooperative adsorption.

AuNP-PEG(2k) shows a similar 'DK value as

citrate-AuNPs and AuNP-PEG(5k) shows no BSA

binding, which indicates that PEG chain length

matters in the BSA binding and longer PEG chain

length prohibited BSA adsorption.

The same experiments were done for TRF on

AuNPs (Figure 3) and similar results were observed.

No TRF adsorption was observed on AuNP-PEG(2k)

and AuNP-PEG(5k). More than 10nm diameter

increase was observed for AuNPs with other four

surface modifications. For citrate-AuNPs and

AuNP-COO-, the nanoparticles-protein interaction

is stronger (smaller 'DK ) for TRF than that for BSA.

The TRF corona thickness on citrate-AuNPs and

AuNP-COO- is ~6-7 nm measured by DLS,

suggesting the monolayer of TRF (hydrodynamic

diameter of TRF ~7 nm[16,24]). For TRF adsorption

on AuNP-Cys, the diameter increased by 60 nm at

high TRF concentrations. The surface of AuNP-Cys

has both –NH3+ and –COO- groups, which could

favor multiple hydrogen bonds or electrostatic

interactions between AuNPs and TRF, and then

accounts for aggregation.

FIB adsorption on AuNPs was also investigated by

DLS (Figure 4). No FIB adsorption was observed on

AuNP-PEG(2k) and AuNP-PEG(5k). Large

aggregation was observed for citrate-AuNPs,

AuNP-COO- and AuNP-Cys (hydrodynamic

diameter up to several hundreds to one thousand

nanometers. This is consistent with the fact that FIB

is easy to aggregate after structure changing

because of knob-hole interactions between different

motives of FIB[25].

At last, we did transmission electron microscopic

(TEM) imaging on the protein coronas by drying

the protein-AuNPs solution on TEM grids (Figure

5). For AuNPs without proteins (Figure 5a-e), no

corona coating was observed except that

AuNP-COO- and AuNP-Cys showed a ~1 nm-thick

corona structure (maybe due to the staining of

surface -COOH by uranyl acetate). For BSA coronas

on citrate-AuNPs, AuNP-COO-, AuNP-Cys and

AuNP-PEG(2k), TEM revealed a corona thickness of

~3-5 nm (Figure 5f-i), which was smaller than that

measured by DLS (6-8 nm). One possibility is that

the protein corona in solution consists of two layers

of proteins since usually one layer BSA corona is ~4

nm of thickness visually on AuNPs and FePt

nanoparticles[10,12]. During drying, soft coronas

were dissociated from AuNPs.

For TRF coronas on citrate-AuNPs and AuNP-COO-,

the thickness observed by TEM was also 3-5 nm

(Figure 5k,i), suggesting TRF monolayer (the

physical dimension of TRF is ~4.2×5×7 nm3[26]). The

TEM measured corona thickness was slightly

smaller than that measured by DLS (~6-7 nm)

possibly due to TRF dehydration.

FIB coronas on citrate-AuNPs, AuNP-COO- and

AuNP-Cys were loosely around AuNPs (Figure

3'

[Protein]([Protein]) (0) 1 (2)

[Protein] ( )

n

z z n nD

d d cK

5

5p-r), which was not so dense as BSA and TRF

coronas. This could be because FIB is a line-like

protein and it was not densely packed on the

surface of AuNPs.

For all protein coronas on AuNP-PEG(2k) and

AuNP-PEG(5k) except BSA on AuNP-PEG(2k), no

protein corona was observed under TEM, matching

with DLS results very well. Note that the contrast of

PEG coating was low and it was hard to be

resolved.

Table 1 summarizes the microscopic dissociation

constant 'DK and Hill coefficient n for all measured

protein coronas in our experiments and some

values from references. All measured microscopic

dissociation constants were in the range of ~9-300

nM. And the Hill coefficient was larger than 1

except for BSA adsorption on citrate-AuNPs. The

values were similar to generally measured values

for protein-nanoparticle interaction[9,11,12,14]. For

example, 'DK and n for BSA adsorption on

citrate-AuNPs measured by fluorescence quenching

method[9] is roughly same as our results.

In conclusion, quantitative analysis has been done

for several serum proteins on AuNPs with different

surface modifications (citrate, thioglycolic acid,

cysteine and PEG). The experimental results have

revealed that (1) Surface modification with long

chain PEG (Mw=5k) can prevent protein adsorption;

(2) FIB tends to introduce aggregation of any AuNPs

not modified with PEG; (3) Surface modification

(citrate, carboxy and both carboxy and amine

modifications) have profound effects on protein

coronas.

Methods

Citrate stabilized AuNPs (40 nm, 9×1010/mL) were

purchased from BBI (England). Ligand exchange was

done at room temperature for 1 h at pH=7.4

[4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid

(HEPES) buffer, 5 mM]. The concentrations of

thioglycolic acid (product#528056, Sigma-Aldrich),

cysteine (product#W326305, Sigma-aldrich),

HS(CH2CH2O)nCH3 (Mw=2k) (Shanghai Seebio

Biotech) and HS(CH2CH2O)nCH3 (Mw=5k)

(Shanghai Seebio Biotech) used in ligand exchange

experiments were 100 μM. Dialysis (MD34, 8-14 kD,

Millipore) was conducted to remove excess ligands.

AuNPs were mixed with BSA, TRF or FIB solution at

room temperature for 30 min and then DLS

characterization was conducted. Final AuNP

concentration was 4.5×108/mL and HEPES buffer was

maintained at 5 mM (pH=7.4). DLS was conducted

on a Malvern zetasizer nano ZS at 25 oC. For a given

surface modification with a certain protein

concentration, three samples were measured and

each sample was measured at least for three times.

Cumulative model was used to fit DLS correlation

curve to get hydrodynamic diameter of nanoparticles.

AuNPs-protein solution (0.5 μM of proteins) was

drop-dried on copper grid and then stained with

uranyl acetate (Zhongjingkeyi Technology Co., Ltd.,

Part# GS02625, saturated aqueous solution) for TEM

imaging. TEM imaging was done on a FEI tecnai at

an operation voltage of 200 kV. UV-Vis was done on

a Lambda 950. All chemicals were purchased from

Sigma Aldrich unless otherwise specified. In all

experiments, DI water (>18 MΩ, 0.2 μm membrane

filtered, Millipore) was used.

Acknowledgements

This work was supported by 973 program (No.

2011CB932803).

Electronic Supplementary Material: Polydispersion

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8

400 500 600 700 8000.0

0.1

0.2

0.3

Ab

so

rpti

on

(a

. u.)

Wavelength (nm)100 101 102 103 104 105

0.0

0.2

0.4

0.6

0.8

1.0 Citrate-AuNPs (dz=43nm)

AuNP-COO- (dz=43nm)

AuNP-Cys (dz=44nm)

AuNP-PEG(2k) (dz=45nm)

AuNP-PEG(5k) (dz=50nm)

g(2

) (

Lag time (s)

Citrate-AuNPs

AuNP-COO-

AuNP-Cys

32 36 40 44 480

20

40

60

Co

un

ts

Diameter (nm)

38.6±3.0 nm

a)

b)

c)

d) e)

100 nm

20 nm

Au Au

Citrate-AuNPs AuNP-COO-

Au

AuNP-Cys

HSCH2COOH

HSCH2CH(NH2)COOH(cysteine)

HSCH2CH2O)nCH3

Au

AuNP-PEG (Mw=2k, 5k)

S

O

n

CH3

AuNP-PEG(2k)

AuNP-PEG(5k)

Figure 1 (a) Typical TEM image of as-bought citrate-stabilized AuNPs. The inset shows an enlarged image. (b) Diameter distribution

of as-bought AuNPs measured by TEM. Totally 200 AuNPs were counted.(c) Schematic illustration of ligand exchange to prepare

AuNP-COO-, AuNP-Cys and AuNP-PEG (2k, 5k). (d) DLS correlation curves and (e) UV-Vis spectra of AuNPs with different

surface modifications.

9

1 10 100 10000.0

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Lag timeS

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1 10 100 10000.0

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g(2

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0.5 5 50 5005000

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10

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0.5 5 50 5005000

0

10

20

dz (

nm)

cBSA

(nM)0.5 5 50 5005000

0

10

20

cBSA

(nM)

dz (

nm

)

=266±120 nMn=0.75±0.17

= 71±32 nMn=3.8±1.7

0.5 5 50 5005000

0

10

20

cBSA

(nM)

d z (

nm)

= 46±14 nMn=1.8±0.9

0.5 5 50 5005000

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dz (

nm)

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(nM)

= 226±54 nMn=1.3±0.2

a) b) c) d)

1 10 100 10000.0

0.2

0.4

0.6

0.8

1.0

0nM 5nM 50nM 500nM

g(2) (

)-1

Lag times

Citate-AuNPs-BSA e)

Figure 2 DLS correlation curves and diameter changes of (a) citrate-AuNPs, (b) AuNP-COO-, (c) AuNP-Cys, (d) AuNP-PEG(2k)

and (e) AuNP-PEG(5k) at different BSA concentrations. 'DK and n in lower panels are fitting parameters using euqation 2 in the

maintext.

10

0.5 5 50 5005000

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)

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dz

(nm

)

= 55±15 nMn=1.4±0.5

a) b) c) d)

= 9.5±3.6 nMn=1.3±0.5

= 150±28 nMn=3.4±1.1

e)

1 10 100 10000.0

0.2

0.4

0.6

0.8

1.0

0nM 5nM 50nM 500nM

Lag timeS

g(2) (

)-1

Citrate-AuNP-TRF

1 10 100 10000.0

0.2

0.4

0.6

0.8

1.0

0nM 5nM 50nM 500nM

Lag timeS

g(2) (

)-1

AuNP-COO--TRF

1 10 100 10000.0

0.2

0.4

0.6

0.8

1.0

0nM 50nM 100nM 500nM

Lag timeS

g(2) (

)-1

AuNP-Cys-TRF

1 10 100 10000.0

0.2

0.4

0.6

0.8

1.0

0nM 5nM 50nM 500nM

Lag timeS

g(2) (

)-1

AuNP-PEG(5k)-TRF

0.5 5 50 5005000

0

10

20

dz (

nm)

cTRF

(nM)

Figure 3 DLS correlation curves and diameter changes of (a) citrate-AuNPs, (b) AuNP-COO-, (c) AuNP-Cys, (d) AuNP-PEG(2k)

and (e) AuNP-PEG(5k) at different TRF concentrations. 'DK and n in lower panels are fitting parameters using euqation 2 in the

maintext.

11

0.5 5 50 5005000

-5

0

5

10

cFIB

(nM)

dz (

nm)

0.5 5 50 5005000

-5

0

5

10

cFIB

(nM)

dz (

nm

)

a) b) c) d) e)

100 101 102 103 104 1050.0

0.2

0.4

0.6

0.8

1.0

1.2

0nM 5nM 50nM 500nM

Lag timeS

g(2) (

)-1

Citate-AuNP-FIB

100 101 102 103 104 1050.0

0.2

0.4

0.6

0.8

1.0

1.2

0nM 5nM 50nM 500nM

Lag timeS

g(2) (

)-1

AuNP-COO--FIB

100 101 102 103 104 1050.0

0.2

0.4

0.6

0.8

1.0

0nM 5nM 50nM 500nM

Lag timeS

g(2) (

)-1

AuNP-Cys-FIB

1 10 100 10000.0

0.2

0.4

0.6

0.8

1.0

0nM 5nM 50nM 500nM

Lag timeS

g(2) (

)-1

AuNP-PEG(2k)-FIB

1 10 100 10000.0

0.2

0.4

0.6

0.8

1.0

0nM 5nM 50nM 500nM

Lag timeS

g(2) (

)-1

AuNP-PEG(5k)-FIB

0.5 5 50 5005000

0

200

400

600

cFIB

(nM)

dz (

nm)

0.5 5 50 50050000

500

1000

1500

cFIB

(nM)

dz (

nm)

0.5 5 50 5005000

0

100

200

300

400

cFIB

(nM)

dz (

nm

)

Aggregation Aggregation Aggregation

Figure 4 DLS correlation curves and diameter changes of (a) citrate-AuNPs, (b) AuNP-COO-, (c) AuNP-Cys, (d) AuNP-PEG(2k) and

(e) AuNP-PEG(5k) at different FIB concentrations. Lines in lower panels are guidance for the eyes.

12

AuNP-PEG(2k)-BSA

AuNP-PEG(5k)-TRF

AuNP-COO--BSAf) g) h)

20 nm 20 nm 20 nm

Citrate-AuNPs-BSA AuNP-Cys-BSA i) AuNP-PEG(5k)-BSA

20 nm

k) l) m)AuNP-COO--TRFCitrate-AuNPs-TRF AuNP-Cys-TRF n) o)AuNP-PEG(2k)-TRF

Citrate-AuNPs-FIB AuNP-PEG(2k)-FIB AuNP-PEG(5k)-FIBp) s) t)

20 nm

20 nm 20 nm 20 nm 20 nm20 nm

j)

q) AuNP-COO--FIB r) AuNP-Cys-FIB

20 nm 20 nm 20 nm 20 nm20 nm

Citrate-AuNPs AuNP-PEG(2k)AuNP-COO- AuNP-Cys AuNP-PEG(5k)a) b) c) d) e)

20 nm 20 nm 20 nm 20 nm20 nm

Figure 5 Typical TEM images of as-prepared AuNPs (a-e) and protein coronas on different AuNPs (f-t).

13

Table 1 Summary of measured microscopic dissociation constant 'DK and Hill coefficient n for BSA and TRF adsorption on

citrate-AuNPs, AuNP-COO- and AuNP-Cys.

BSA TRF FIB

'DK (nM) n '

DK (nM) n 'DK (nM) n

Citrate-AuNPs 266±120

Ref9: 301±51

Ref10: 2.56±0.50×105

Ref7: 2.0×103

0.75±0.17

Ref9: 0.81±0.03

Ref10: 0.4±0.1

55±15 1.4±0.5 -

(Aggregation)

-

AuNP-COO- 71±32 3.8±1.7 9.5±3.6 1.3±0.5 -

(Aggregation)

-

AuNP-Cys 46±14 1.8±0.9 150±28

(Aggregation)

3.4±1.1

-

(Aggregation)

-

AuNP-PEG(2k) 226±54 1.3±0.2 -

(no binding)

- -

(no binding)

-

AuNP-PEG(5k) -

(no binding)

-

-

(no binding)

-

-

(no binding)

-

Notes: In Ref. 9, human serum albumin (HSA) and AuNPs with 30nm diameter were used. In Ref. 10, AuNPs with 56nm diameter

were used and DLS was measured immediately after AuNPs and BSA mixing. In Ref. 7, AuNPs with 60nm diameter were used and

Langmuir model fitting was used (equal to n fixed at 1 in Hill equation.)