Supplementary Information SUPPLEMENTARY INFORMATION · S5 where K is the Scherrer constant, is the...

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2012.220

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 1S1

Supplementary Information

Electrostatic Assembly of Binary Nanoparticle Superlattices

Using Protein Cages

Mauri A. Kostiainen*, Panu Hiekkataipale, Ari Laiho, Vincent Lemieux, Jani Seitsonen, Janne

Ruokolainen, Pierpaolo Ceci

Contents

1. Materials and Methods .......................................................................................................... 2

Preparation and Characterisation of Nanoparticles .................................................................... 2

Small Angle X-ray Scattering (SAXS) ...................................................................................... 4

Cryo Transmission Electron Microscopy (Cryo-TEM) ............................................................. 5

Cryo Electron Tomography (Cryo-ET) ...................................................................................... 5

Superconducting Quantum Interference Device (SQUID) Measurements ................................ 6

Magnetic Resonance Imaging (MRI) ......................................................................................... 6

Dynamic Light Scattering (DLS) and Electrophoretic mobility ................................................ 6

Agarose Gel Electrophoresis Mobility Shift Assay (EMSA) .................................................... 7

2. Additional SAXS Data .......................................................................................................... 7

3. Additional TEM Images ...................................................................................................... 11

4. Binding of AuNPs on the Negative Patches of Protein Cages ............................................ 13

5. Magnetic Properties and Free-Standing Superlattices......................................................... 14

6. MR Imaging ........................................................................................................................ 16

7. DLS and Electrophoretic Mobility Data ............................................................................. 17

8. Agarose Gel Electrophoresis Mobility Shift Assay Data .................................................... 19

9. Supplementary Information References .............................................................................. 19

© 2012 Macmillan Publishers Limited. All rights reserved.

S2

1. Materials and Methods

Preparation and Characterisation of Nanoparticles

Gold nanoparticles (AuNPs) were prepared according to the biphasic method developed by Brust

and SchiffrinS1,S2

and the cationic ligand (1)

was prepared according to a procedure

developed by Rotello and coworkersS3

. The

preparation of cationic AuNPs is described here

briefly for clarity.

Pentane-thiol AuNPs: A solution of hydrogen tetrachloroaurate trihydrate (310 mg, 0.79 mmol,

1 equiv.) in deionised water (25 mL) was added to a solution of tetraoctylammonium bromide (1.08

g, 2.0 mmol, 2.5 equiv.) in toluene (79 mL) in a 125 mL Erlenmeyer flask containing a magnetic

stirring bar. The biphasic mixture was vigorously stirred until all the tetrachloroaurate had

transferred into the organic phase, giving a deep orange solution and a clear aqueous solution. After

10 minutes, the organic layer was isolated using a separatory funnel and transferred to a 250 mL

Erlenmeyer flask equipped with a magnetic stirring bar. A solution of 1-pentanethiol (24.3 µL, 20.5

mg, 0.2 mmol, 0.25 equiv.) in toluene (1 mL) was added to the flask. The resulting mixture was

vigorously stirred for 10 minutes, after which a solution of a sodium borohydride (300 mg, 7.9

mmol, 10 equiv.) in deionised water (25 mL) was added in less than 10 seconds. The colour of the

organic layer became immediately dark brown. The reduction was very exothermic. The solution

was stirred overnight at ambient temperature. The organic phase was isolated and the solvent was

removed using a rotary evaporator with the water bath temperature set below 35 °C. The dark solid

was suspended in ethanol (30 mL), centrifuged, and decanted. This washing procedure was repeated

three times. The pentane-thiol gold nanoparticles were then dried under high vacuum.

1H NMR: (CDCl3, 400 MHz) δ 2.5 – 0.5 (broad). TEM: Particle (Au core) diameter 2-3 nm.

Cationic AuNPs: A solution of compound 1 (120 mg, 0.28 mmol) in water (5 mL) was added to

a stirred solution of pentane-thiol AuNPs (30 mg) in dichloromethane (5 mL). The gold

nanoparticles transferred spontaneously from the dichloromethane phase to the aqueous phase. The

mixture was stirred for 1 hour. The solvent was removed under vacuum using a rotary evaporator.

The residue was dissolved in a minimum of deionised water and placed in a dialysis bag. The

solution was dialysed against deionised water for 48 hours. The cationic nanoparticles were isolated

as a brown solid after lyophilisation.

1

OO

OO

N+

SH


S3

1H NMR: (D2O, 400 MHz) δ 0.31–1.59 (br, 18H); 3.05 (s, 9H); 3.26–3.78 (br, 18H); 3.80–3.91 (br,

2H). TEM: Particle (Au core) diameter 2.55 0.49 nm.

The number density of AuNPs was estimated to be ~ 78 1015

NPs mg-1

based on known

absorption at 450 nm. It must be noted that this number and the particle number ratios presented in

Fig. 2a can only be treated as crude estimates since the number density analysis of small (DAu < 3

nm) AuNPs is challengingS4

and easily leads to large errors. However, the mass ratios presented in

Fig. 2a can be accurately measured.

Figure S1. Characterisation of cationic gold nanoparticles

with TEM. Particle (Au core diameter) size distribution of the

cationic AuNPs with a Gaussian fit. TEM image of the AuNPs

(inset), scale bar 10 nm.

CCMV particles were grown and isolated from California black-eye beans as reported

previouslyS5,S6

. apoFT and MF with approximately 3500 encapsulated Fe atoms were purchased

from Molecular Links Rome (MoLiRom, www.molirom.com). MF was cationised at room

temperature following the Thermo Scientific manufacturer's protocol for 1-ethyl-3-[3-

dimethylaminopropyl]carbodiimide hydrochloride (EDC) coupling (subject to minor

modifications). Briefly, the concentration of the MF solution was 2 mg mL-1

in PBS buffer at pH

6.5. To this solution, EDC (4 mM), sulfo-N-hydroxysulfosuccinimide (sulfo-NHS, 10 mM) and

NN-dimethyl-1,3-propanediamine (DMPA, 50 mM) were added sequentially and the pH was

maintained at 6.5 by additions of 1 N NaOH with continuous stirring. EDC and sulfo-NHS were

added directly as powder, while DMPA was prepared as a 0.8 M solution at pH 6.5. After 10

minutes, the pH was raised to 7.3 with 1 M Hepes buffer pH 7.5 and the reaction was allowed to

proceed for 1 hour at room temperature. Finally, the ferritin solution was filtered and exchanged 4–

5 times with MQ-H2O using 30 kDa Amicon Ultra-15 centrifugal filter devices (Millipore) to

remove the excess reagents. The cationised sample was analysed by agarose gel electrophoresis (pH

8.5), which showed reduced mobility. Finally the sample was sterile-filtered and stored at 4 °C.

0 1 2 3 4 5 6 7 8 9 100

20

40

60

80

µ = 2.55 nm = 0.49 nm

Diameter (nm)

Coun

ts


S4

Summary of nanoparticle sizes is presented in Fig. S2.

Figure S2. Size of the nanoparticles used in this study. a, Table summarising the nanoparticle sizes observed by

TEM and DLS. b, Diameter of the AuNPs with the cationic ligand included was estimated from TEM images where the

AuNPs formed small arrays. These measurements assume 2D ordering.

Small Angle X-ray Scattering (SAXS)

Binary superlattices were prepared by slowly mixing together 5 L of CCMV (10 mg mL-1

0.1 M

NaAc, pH 3-6) or apoFT / MF solution (10 mg mL-1

0.1 M HEPES pH 7.5 or 0.1 M NaAc, pH 3.7),

1.25 L of NaCl, MgCl2 or KCl (0-2 M) to adjust the ionic strength (effect of the buffer to Debye

length was omitted) and appropriate volume of AuNP solution (120 mg mL-1

in MQ-H2O). 10 L

of the sample was sealed in a metal or plastic holder between two Kapton films to yield a sample

with liquid thickness of approximately 0.9 mm. The structural periodicities were measured by using

a rotating anode Bruker Microstar microfocus X-ray source (Cu K radiation, λ = 1.54 Å) with

Montel collimating optics. The beam was further collimated with four sets of slits (JJ X-ray),

resulting in a beam of approximately 1 × 1 mm at the sample position. The distance between the

sample and the Hi-Star 2D area detector (Bruker) was 1.59 m. One-dimensional SAXS data were

obtained by azimuthally averaging the 2D scattering data. The magnitude of the scattering vector is

given by:

eq. (S1)

where 2θ is the scattering angle. Calculated SAXS curves in Fig. 2 and 4 were obtained by

PowderCell software.

The relative sample crystallinity (crystal domain size) was estimated from the full-width at half

maximum (FWHM) of the highest intensity reflection in the diffraction pattern using the Scherrer

equationS7

:

eq. (S2)

CCMV apoFT MF AuNP

TEM DLS TEM DLS TEM DLS TEM DLS

Dcore (nm) - - - - 6.1 1.0 - 2.6 0.5 -

Douter (nm) 28.0 1.0 28.3 12.5 0.4 12.5 12.2 0.8 12.4 8.1 0.4 9.5

24.3 nm

a) b)


S5

where K is the Scherrer constant, is the x-ray wavelength and B is the integral breadth. The

presence of lattice imperfections, sub-grain boundaries lattice strains and instrumental factors also

contribute to the broadening of the X-ray reflections and decrease the apparent domain size

(B2

sample = B2

measured - B2

instrument). Here we do not use correction for instrumental factors, which

would amplify the difference between the amorphous and crystalline samples. All the samples were

measured with the same instrument using identical parameters. The estimated value is therefore the

lower limit of the single crystal domain size. The relative degree of crystallinity () of a given

sample N has no unit and was calculated as:

eq. (S3)

Since cosB in the studied q-vector range varies only between 0.9990.983 and the highest intensity

reflection remains almost constant, eq. S3 can also be reliably approximated as:

eq. (S4)

Cryo Transmission Electron Microscopy (Cryo-TEM)

Cryo-TEM samples were prepared in a similar manner as for the SAXS measurements (pH 5) and

diluted by a factor of 1:50. Prior to sample deposition the TEM grids (Quantifoil R 3.5/1, holey

carbon film, Cu 200 mesh) were treated with Gatan Solarus 950 plasma system. 3 L of sample was

placed on the grid, which was consecutively blotted for 1-2 s (100 % relative humidity, -2 mm blot

offset), with FEI Vitrobot Mk3 followed by immediate vitrification with a mixture of liquid ethane

and propane (~1:1) at 180 °C. Vitrified samples were cryo-transferred to the microscope. Images

(4096 4096 pixels) were obtained with a JEOL JEM-3200 FSC field emission cryo electron

microscope operating at a 300 kV accelerating voltage and specimen temperature of 86 K. Images

were processed with Gatan DigitalMicrographTM

and ImageJ softwares.

Cryo Electron Tomography (Cryo-ET)

Single-axis tilt series images at 2 increment were acquired in low-dose mode with serialEM

softwareS8

. Total electron dose during record phase was ~50 e/Å2. The tilt series images were

aligned and 3D reconstructed with IMOD/Etomo program suite. 3D tomographic reconstructions

were finally visualised and rendered with Chimera.


S6

Superconducting Quantum Interference Device (SQUID) Measurements

Magnetometry measurements were performed with a Quantum Design Magnetic Properties

Measurement System (MPMS XL-5). MF-AuNP samples (90 µL, containing MF 0.05 mg mL-1

)

were sealed inside polycarbonate capsules and rapidly quenched with liquid N2. Frozen samples

were loaded to the MPMS operating at 200 K to minimise evaporation. Diamagnetic background

was subtracted from the raw data.

Magnetic Resonance Imaging (MRI)

MRI samples were prepared in a similar manner as the SAXS samples, diluted and embedded into

agarose gel to yield the given sample concentrations, a final gel concentration of 1 % and 200 µL

volume. Samples were finally loaded onto a standard 96-well plate. Magnetic resonance imaging

(MRI) experiments were performed on a 3-T Magnetom Skyra whole-body scanner (Siemens,

Erlangen, Germany), using a standard 20-channel head-neck coil. Spin echo pulse sequence with

eight different echo times (TE = 12-120 ms) and a constant repetition time (TR = 2000 ms) was used

to measure spin-spin relaxation times (T2). Spin-lattice relaxation times (T1) were measured by

using inversion recovery technique with seven different inversion times (TI = 100-4910 ms,

TR = 5000 ms and TE = 13 ms). Coronal images were acquired by using a single slice of thickness

2.0 mm with a matrix of 256 x 256 and a field of view of 50 x 50 mm (T2) or 60 x 60 mm (T1).

Mean signal intensities of each sample within a circular region-of-interest (ROI) were plotted

against TE and TI and fitted to an exponential function to extract T2 and T1, respectively. Finally,

relaxation rates 1/T2 and 1/T1 were plotted against the concentration of iron (Fe) in the samples and

the transverse (r1) and longitudinal relaxivities (r2), respectively, were calculated from the slope of

the fitted (linear regression) lines. Two and three-dimensional MR images were acquired using

standard spin-echo and Siemens SPACE pulse sequences (3D turbo spin echo with TE = 409 ms and

TR = 3200 ms), respectively.

Concentration of iron (Fe) was analysed by using graphite furnace atomic absorption

spectrometry (GFAAS) at the trace element laboratory located within the University of Oulu.

Dynamic Light Scattering (DLS) and Electrophoretic mobility

Experiments were carried out at 25 C with a Zetasizer Nano ZS from (Malvern Instruments)

equipped with a 4 mW He-Ne ion laser at a wavelength of 633 nm and an Avalanche photodiode

detector at an angle of 173°. DLS results are the average of at least six 10 s runs. All CCMV


S7

apoFT / MF samples (2040 mg L-1

) were prepared in filtered buffer solutions (10 mM NaAc, pH

5) and (10 mM HEPES, pH 7), respectively. The protein cage solutions (0.5 mL,) were titrated with

an aqueous AuNP solution (0.00110 mg mL-1

depending on the titration). The added AuNP

solution did not exceed 5% of the total volume, therefore no corrections were made for sample

dilution. The samples were thoroughly mixed and allowed to equilibrate for at least five minutes.

Agarose Gel Electrophoresis Mobility Shift Assay (EMSA)

Agarose gels were prepared from a solution of 1.2 % agarose in dilute acetate buffer (10 mM NaAc,

pH 5) and stained with 10 μL of ethidium bromide solution (10 mg mL-1

). Samples (16 µL)

contained CCMV (78 mg L-1

) and the AuNPs in various ratios. 1 μL of 6X nucleic acid loading dye

(Fermentas) was added to each sample. A sample of the solution (15 μL) was run at 100 V for 30

min with 10 mM NaAc, pH 5 as the running buffer. Gels were imaged with Bio-Rad Gel DocTM

EZ imaging system and analysed with Image Lab 3.0 build 11 software.

2. Additional SAXS Data

Fig. S3 presents the raw SAXS data at different AuNP/CCMC ratios, screening lengths and pH

values used to plot the Fig. 2 of the main article. Interestingly the CCMV and AuNPs adopt only

one type (AB8fcc

) of crystalline particle arrangement. Section 4 contains additional discussion on

how the patchiness of the protein cage directs the superlattice formation.


S8

Figure S3. Raw SAXS data of CCMV-AuNP samples. a-d, Integrated 1D curves (left) and 2D SAXS patterns (right)

of CCMV-AuNP samples at different NaCl concentrations and with different mAuNP/mCCMV ratios: a) 0.5, b) 1, c) 1.5, d)

2. e-j, Integrated 1D curves CCMV-AuNP samples at different pH values and NaCl concentrations. The mAuNP/mCCMV

ratio for the pH series was 0.75.

The effect of the electrolyte type on the superlattice formation was studied by using MgCl2,

NaCl and KCl. Figure S4 presents integrated 1D SAXS curves for the different electrolytes. The

same behaviour as presented in Figure 2 is observed, irrespective of the electrolyte identity. At low

electrolyte concentrations gel-like aggregates are observed. The superlattice formation is efficient

0.02 0.07 0.12

0306090120160200250300

0.02 0.07 0.12

0306090120160200250300

0.02 0.07 0.12

0306090120160200250300

0.02 0.07 0.12

0306090120160200250300

0 30 60

90 120 160

200 250 300

0 30 60

90 120 160

200 250 300

0 30 60

90 120 160

200 250 300

0 30 60

90 120 160

200 250 300

I(q) [a

.u]

q (Å-1)

I(q) [a

.u]

q (Å-1)

I(q) [a

.u]

q (Å-1)

I(q) [a

.u]

q (Å-1)

a b

c d

mAuNP/mCCMV = 0.5

c [NaCl] (mM)

mAuNP/mCCMV = 1

mAuNP/mCCMV = 1.5 mAuNP/mCCMV = 2

c [NaCl] (mM)

c [NaCl] (mM) c [NaCl] (mM)

0.02 0.07 0.12

0

30

60

90

120

160

0.02 0.07 0.12

0

30

60

90

120

160

0.02 0.07 0.12

0

30

60

90

120

160

0.02 0.07 0.12

0

30

60

90

120

160

0.02 0.07 0.12

0

30

60

90

120

160

0.02 0.07 0.12

0

30

60

90

120

160

200

I(q) [a

.u]

q (Å-1)

I(q) [a

.u]

q (Å-1)

I(q) [a

.u]

q (Å-1)

I(q) [a

.u]

q (Å-1)

I(q) [a

.u]

q (Å-1)

I(q) [a

.u]

q (Å-1)

c [NaCl] (mM) c [NaCl] (mM) c [NaCl] (mM)

c [NaCl] (mM) c [NaCl] (mM) c [NaCl] (mM)

pH 3 pH 3.4 pH 3.7

pH 4.5 pH 5 pH 6

e f g

h i j


S9

when -1

~ 1.20.9 and at shorter screening lengths only free particles are observed. Importantly,

the region where the superlattice formation takes place is the same also with divalent MgCl2 in

terms of screening length, which is achieved with lower molar MgCl2 concentrations than with

monovalent electrolytes, such as NaCl.

Figure S4. SAXS data of CCMV-AuNP samples with different electrolytes. a-c, Integrated 1D curves of CCMV-

AuNP samples at different concentrations of a, MgCl2, b, NaCl and c, KCl.

Similarly to the CCMV-AuNP superlattices, the formation of apoFT-AuNP superlattices is pH

sensitive. Adjusting the pH to 3.7 (below the pI of apoFT) leads to conditions where the superlattice

formation is prevented. Figure S5a shows 1D SAXS curves for apoFT at different NaCl

concentrations at pH 3.7 which indicate the absence of superlattices. At pH 7.5, but otherwise

identical conditions, the superlattice formation is efficient. Furthermore, if the protein cage of MF is

chemically cationised with NN-dimethyl-1,3-propanediamine the formation of superlattices can be

prevented even at neutral pH (Figure S5b). Finally, if small cationic ligands are used instead of the

AuNPs, close packed fcc arrangement of MF can be achieved (see main article ref. 24).

Figure S5. SAXS data of FT-AuNP samples. a, Formation of apoFT-AuNP superlattices is prevented at pH 3.7,

which is below the pI of FT. b, Chemically cationised MF cannot form superlattices with AuNPs at neutral pH.

Unit cell details obtained from SAXS measurements for (CCMV-AuNP8)fcc

, (aFT-AuNP)sc

and

(MF-AuNP)sc

superlattices are summarised in Table S1.

0 0.05 0.1 0.15

>3 nm

1.8 nm

1.2 nm

1.0 nm

0.9 nm

0.8 nm

0 0.05 0.1 0.15

>2 nm

1.2 nm

0.88 nm

0.72 nm

0.62 nm

0 0.05 0.1 0.15

>3 nm

1.8 nm

1.2 nm

1.0 nm

0.9 nm

0.8 nm

I(q) [a

.u]

q (Å-1)

I(q) [a

.u]

q (Å-1)

I(q) [a

.u]

q (Å-1)

MgCl2 NaCl KCla b c

-1 -1 -1

0 0.05 0.1 0.15

0 mM NaCl, pH 3.7

30 mM NaCl, pH 3.7

60 mM NaCl, pH 3.7

90 mM NaCl, pH 3.7

90 mM NaCl, pH 7.5

I(q) q

2[a

.u]

q (Å-1)

pH 3.7

pH 7.5

0 0.05 0.1 0.15

cationised MF, 0 mM NaClcationised MF, 30 mM NaClcationised MF, 60 mM NaClcationised MF, 90 mM NaClnative MF, 90 mM NaCl

I(q) q

2[a

.u]

q (Å-1)

cationised MF

native MF

a b


S10

Table S1. Unit cell details obtained by SAXS.

(CCMV-AuNP8)fcc

(aFT-AuNP)sc

(MF-AuNP)sc

Bravais lattice face-centred cubic simple cubic simple cubic

Space group (number) Fm m (225) Pm m (221) Pm m (221)

Unit cell size

(lattice parameter a) 40.4 nm 12.4 nm 12.9 nm

Protein cage

centre-to-centre distance 28.6 nm 12.4 nm 12.9 nm

Particles in unit cell 36 (4 CCMV, 32 AuNP) 2 (1 MF, 1 AuNP) 2 (1 MF, 1 AuNP)

Primitive vectors

Basis vectors x,y,z

(Protein cage)

(AuNP)

A1 = ½ aY + ½ aZ

A2 = ½ aX + ½ aZ

A3 = ½ aX + ½ aY

B1 = (0A1, 0A2, 0A3)

B2 = (0.35A1, 0.35A2, 0.35A3)

A1 = aX

A2 = aY

A3 = aZ

B1 = (0A1, 0A2, 0A3)

B2 = (0.5A1, 0.5A2, 0.5A3)

A1 = aX

A2 = aY

A3 = aZ

B1 = (0A1, 0A2, 0A3)

B2 = (0.5A1, 0.5A2, 0.5A3)

Unit cell image

CCMV: blue spheres

AuNP: yellow spheres

apoFT: transparent light

red spheres

MF: solid red spheres

(sphere diameter for

(CCMV-AuNP8)fcc

has

been reduced for clarity)

Illustration of the (CCMV-AuNP8)fcc

superlattice. a, Unit cell showing only the simple cubic arrangement of eight

AuNPs located at the octahedral void in the centre of the unit cell. b, Unit cell showing only the 32 AuNPs. c, Unit

cell with 9 CCMV particles (top five particles omitted) and AuNPs. d, Unit cell with all CCMV particles and AuNPs.

a db c


S11

3. Additional TEM Images

Figure S6. Low-Magnification cryo-TEM images of (CCMV-AuNP8)fcc

superlattices. a, Typical cryo-TEM view

showing multiple 3D superlattices from different orientations. b, (CCMV-AuNP8)fcc

superlattice viewed along the [111]

zone axes.

Figure S7. Cryo-TEM images of MF-AuNP samples. a, Cryo-TEM image showing amorphous gel-like aggregates of

MF-AuNP samples prepared in the absence of NaCl. b, Low-magnification image of 3D (MF-AuNP)sc

superlattices that

form in the presence of 90 mM NaCl (corresponding to -1 = 1 nm) (left image). Higher magnification of small

superlattices viewed from different orientations (right image). c, At 200 mM NaCl concentration the electrostatic

attraction between the particles is reduced and consequently only free individual particles are observed.

a b

a b

c


S12

Figure S8. Cryo-TEM images of vitrified aqueous solution containing MF-AuNP superlattices. View along a,

[100], b, [110] and c, [111] projection axes. Each panel contains a cryo-TEM image (scale bar 25 nm), image Fourier

transform (top), inverse Fourier transform calculated with selected Fourier components (middle) and a unit cell viewed

along the given projection axes (bottom).

Figure S9. 3D reconstruction of protein cage-nanoparticle lattices obtained by electron tomography. Large view

on: a, (CCMV-AuNP8)fcc

superlattice and b, (MF-AuNP)sc

superlattice. c, Three dimensional electron density maps

obtained by cryo-electron tomography. (CCMV-AuNP8)fcc

superlattice viewed along the [100] (left image) and [110]

(right image) zone axes. Unit cells are outlined in cyan. d, The respective electron density maps as shown in a mapped

with the CCMV (blue) and Au (yellow) nanoparticles.

a b c

a b

c d[100] [110] [100] [110]


S13

4. Binding of AuNPs on the Negative Patches of Protein Cages

The negative charge density on the CCMV and FT is not homogenously distributed, but located in

patches. CCMV consists 180 identical protein subunits that form 12 pentamers and 20 hexamers to

create a T = 3 capsid with an overall icosahedral symmetry. Large negatively charged patches are

located around the 60 pores present in the centre of the three-fold quasi-icosahedral rotation axes.

The pore area is known to bind multivalent cationsS9

. FT cage consists of 24 identical protein

subunits and has octahedral symmetry created by cubic point group 432. The enclosing polyhedron

has 24 vertices, 48 edges and 26 faces of which 6 are squares, 8 are equilateral triangles and 12

rectangles.S10

Also the FT cage has negative sites located around the eight pores along the threefold

symmetry axes.

On the CCMV capsid, the distance between the adjacent negative patches is approximately 6 nm,

which indicates that two AuNPs with Dh ~ 8 nm are unable to bind to adjacent patches due to steric

hindrance and electrostatic repulsion between the AuNPs. However, when AuNPs are mapped to

the negative patches so that one empty patch is placed between the AuNPs, altogether 24 AuNPs

can be placed in contact with one capsid. Figure S10a frame and surface presentations show a

schematic presentation where the AuNPs bind viewed from different orientations. Below them is a

comparison to the experimental data. Cryogenic-TEM tomography data shows one virus particle

and the position of the 24 AuNPs that are in contact with the capsid and the TEM images shows the

lattice viewed along the respective projection axes. The experimental data matches with the model

and explains that the patchiness of the capsid may direct the formation of AB8fcc

superlattice and

suppresses the formation of other structures that could be expected, such as AB8hcp

. The formation

of MF-AuNP superlattice is also governed by the location of negative patches. Here the patches are

located on the 8 equilateral triangle faces. Mapping the AuNPs on these faces yields a simple cubic

arrangement, which matches the experimentally observed structural configuration (Figure S10b).


S14

Figure S10. Binding of AuNPs on the surface of protein cages. a, Top left: Crude electrostatic potential of the

CCMV capsid viewed along the two-fold symmetry axis. Top right: Calculated electrostatic surface potential of three

protein subunits surrounding the pore at quasi three-fold axis. Box: Schematic and experimental presentation of one

CCMV binding 24 AuNPs. b, Top left: Crude electrostatic potential of the Pyrococcus furiosus cage viewed along the

three-fold symmetry axis. Top right: Calculated electrostatic potential on the solvent accessible surface of three protein

subunits surrounding the pore at three-fold axis. Box: Schematic and experimental presentation of one MF binding 8

AuNPs. In top images, red and blue colors represent negative and positive electrostatic potential, respectively. Values

range from 0 kBT e-1

(blue) to -9 kBT e-1

(red), where kB = Boltzmann constant, T = absolute temperature, and

e = elementary charge. Electrostatic surface potentials for the CCMV and MF protein trimers were calculated using the

PDB2PQR web portal (http://nbcr-222.ucsd.edu/pdb2pqr_1.8/)S11

.

5. Magnetic Properties and Free-Standing Superlattices

Magnetometry measurements with a superconducting quantum interference device (SQUID) were

carried out to characterise the magnetic properties of the crystalline MF-AuNP assemblies. Fig. S11

shows the magnetisation curve measured at 9 K, which indicates saturation at fields larger than

±0.5 T. At lower temperatures (5 K) clear coercivity (Hc) was observed. Zero-field-cooled (ZFC)

a b


S15

and field cooled (FC) curves using a 10 mT measuring field demonstrates a transition from

ferromagnetic to superparamagnetic state at TB ~ 20 K.

We further demonstrated that the MF-AuNP superlattices can be magnetically separated, purified

and dried to yield dry free-standing superlattices. Such superlattices may have important

implications in designing nanodevices and metamaterialsS12

. In aqueous solution, a mixture of free

AuNP and MF particles at 200 mM NaCl cannot be separated with a permanent magnet (Fig. S11c),

whereas the superlattices (and gel-like aggregates, not shown) are quickly attracted to the magnet

(Fig. S11d). The free-standing superlattices were prepared in a similar manner as the regular SAXS

samples. However, before measurements the superlattices were collected to the side of the vessel

with a magnet and the liquid was removed. The superlattices were then dried under vacuum and

finally measured with SAXS, which showed that the lattices were almost identical to those observed

in solution (Fig. S11e).

Figure S11. Magnetic properties and formation of free-standing MF-AuNP superlattices. a, Hysteresis loop at 9 K

indicates saturation at fields larger than ±0.5 T. b, Temperature dependent zero-field-cooled (ZFC) and field-cooled

(FC) magnetisation using a 10 mT measuring field. Blocking temperature TB ~ 20 K. c, Free MF and Au nanoparticles

in buffered water with 200 mM NaCl are not attracted to a permanent neodymium magnet (B = 0.6 T). d, However,

superlattices of the same nanoparticle components can be separated within 1 min with the same magnet. e, Integrated

1D SAXS curve obtained from the magnetically separated and dried free-standing superlattices shows a CsCl-type

packing (inset: MF, red; AuNP, yellow).

2

6

10

14

18

0 20 40 60 80 100

-1.5

-1

-0.5

0

0.5

1

1.5

-1 -0.5 0 0.5 1

M/M

s

0T (T)

M (

em

u)

10

5

T (K)

0 0.05 0.1 0.15

a = 13.38 nm

(100

)

(110

)(1

11)

(200

)(2

10)

(211

)

(220

)(3

00)

I(q

) q

2[a

.u]

q (Å-1)

ec dfree particles (MF+AuNP) superlattices (MF+AuNP)

c[NaCl] = 90 mMc[NaCl] = 200 mM

B = 0.6 TB = 0.6 T

a b

ZFC

FC


S16

6. MR Imaging

Magnetoferritin can enhance contrast in MR imaging in two ways: by shortening spin-lattice

relaxation time (T1) of protons or by modulating spin–spin relaxation times (T2) of surrounding

protons by enhancing the spin dephasing. In general, iron oxide based materials are efficient T2

contrast agents compared to Gadolinium chelates (Fig. S12a), such as MagnevistS13

. This can be

observed in the measured relaxivity of free MF (r2 = 41 s-1

mM-1

), which is much higher than for

Magnevist (r2 = 4.8 s-1

mM-1

) and in line with previous reports on MF with similar iron oxide core

sizesS14

(it should be noted that exact relaxivity values depend on the field strengthS15

). T2

relaxivities of iron oxide nanoparticles can be further enhanced by clustering multiple nanoparticles

into higher-order complexesS16,S17

. We observed a 37 % increase in the relaxivity r2 = 56 s-1

mM-1

when MF was organised into superlattices. Moreover the relaxivity is further increased

r2 = 76 s-1

mM-1

with the amorphous complexes due to efficient aggregation. An opposite trend can

be observed with T1 relaxation times (Fig. S12b), where increasing aggregation reduces the

relaxivity (free particles: r1 = 1.9 s-1

mM-1

, superlattices: r1 = 1.5 s-1

mM-1

, amorphous:

r1 = 0.8 s-1

mM-1

). The measured relaxivities yield high relaxivity ratios (r2/r1), especially for the

superlattices (r2/r1 = 37) and amorphous complexes (r2/r1 = 95). Taken together, these results

indicate that hierarchical magnetoferritin assemblies can provide contrast enhancement in MR

imaging, but also in applications that are not immediately obvious and are not related to biological

studies, such as fluid dynamicsS18

. A 37 % increase in the r2 relaxivity with the superlattices

compared to free MF particles indicates that the superlattices may, in principle, be utilised as

magnetic resonance switches for the detection of various analytesS19

. Our observation that

superlattices and amorphous aggregates composed of many nanoparticles give higher/lower

transverse/longitudinal relaxivities, respectively, than separate free nanoparticles is consistent with

a model put forward by Gillis and coworkersS20

.


S17

Figure S12. Magnetic resonance imaging. a-b, 1/T2 (spin-spin) (a) and 1/T1 (spin-lattice) (b) relaxation rates of (MF-

AuNP)sc

superlattices (-1 = 1 nm), free MF-AuNP system (-1

= 0.8 nm), amorphous MF-AuNP complex (-1 > 3 nm)

and commercially available MagnevistS21

. c-d, T2 (c) and T1 (d) weighted magnetic resonance images (MRI) of 1 %

agarose gel phantoms containing different amounts of (MF-AuNP)sc

superlattices. e, T2-weighted 2D images of (MF-

AuNP)sc

superlattices (cFe = 3.6 mM) injected into the centre of three 200 L gel phantoms. f, T2-weighted 3D image of

(MF-AuNP)sc

superlattices (cFe = 3.6 mM) injected into the centre of a 20 mL gel phantom.

7. DLS and Electrophoretic Mobility Data

The formation of free particles and AuNP-protein cage assemblies was studied by DLS. The

hydrodynamic diameter (Dh) of AuNPs is 9.5 nm as observed by DLS. This value corresponds well

with the diameter of 8.1 nm nanometres, which was estimated by TEM. Native CCMV capsid has

an outer diameter of 28 nm and, as expected, the diameter of 28.3 nm observed by DLS matches

closely with the expected value. The amorphous gel-like CCMV-AuNP assemblies (region 1 in Fig.

2) form quickly and are not perfectly suited for DLS, which indicates the presence of aggregates

with broad size distribution. In the presence of 90 mM NaCl the assemblies become ordered and

size of crystalline assemblies is observed to set ~1.5 m. Further increase in the salt concentration

TR = 2000 msTE = 40 ms

cFe (mM) T2

a

c

1/T

2(s

-1)

cFe (mM)

0

30

60

90

0 0.25 0.5 0.75 1

MF + AuNP (free particles)MF + AuNP (superlattice)MF + AuNP (amorphous)Magnevist

0

1

2

3

4

0 0.25 0.5 0.75 1

MF + AuNP (free particles)MF + AuNP (superlattice)MF + AuNP (amorphous)Magnevist

1/T

1(s

-1)

cFe (mM)

b

TR = 400 msTE = 10 ms

cFe (mM) T1

d0.06

0.11

0.22

0.45

0.90

0

0.06

0.11

0.22

0.45

0.90

0

r2 = 56 (s-1 mM-1)

r2 = 41 (s-1 mM-1)r2 = 4.8 (s-1 mM-1)

r2 = 76 (s-1 mM-1)

r2 = 1.5 (s-1 mM-1)

r2 = 1.9 (s-1 mM-1)r2 = 4.4 (s-1 mM-1)

r2 = 0.8 (s-1 mM-1)

e f


S18

leads to the decrease in the area of interaction between the particles and consequently the strength

of electrostatic interactions yielding freely dissolved particles. The assembly process is rapid and

takes place within minutes after adding the AuNPs. Electrophoretic mobility measurements indicate

that the CCMV particles are negatively and AuNPs positively charged. Addition of AuNPs to the

CCMV results clearly in complexes, which are less negatively charged than the free CCMV

particles. Similar trends as for the CCMV are observed in the case of MF-AuNP complexes (Fig.

S13d).

Figure S13. Self-assembly of CCMV-AuNP complexes. a, Volume-averaged size distribution of free CCMV,

amorphous gel-like CCMV-AuNP aggregates (region 1), superlattices (region 2), a mixture of free CCMV and AuNPs

at high NaCl concentration (region 3) and free AuNPs. b, Second order autocorrelation functions of the corresponding

curves presented in panel a. c, Electrophoretic mobility measured for free CCMV, CCMV-AuNP aggregates and free

AuNPs. d, Volume-averaged size distribution of free MF, amorphous gel-like MF-AuNP aggregates, crystalline

superlattices, a mixture of free CCMV and AuNPs at high NaCl concentration (region 3). Inset: the corresponding

second-order autocorrelation functions.

0

5

10

15

20

25

30

0.1 1 10 100 1000 10000

MF 25 mg/Lregion 1 (amorphous)region 2 (crystalline)region 3 (free particles)

-15 -5 5 15

CCMV (40 mg/L)

CCMV + AuNP (44 mg/L)

AuNP (80 mg/L)

Mobility (10-4 cm2 V-1 s-1)

0

5

10

15

20

25

30

0.1 1 10 100 1000 10000

CCMV 40 mg/Lregion 1 (amorphous)region 2 (crystalline)region 3 (free particles)AuNPs (80 mg/L)

Dh (nm)

Inte

nsity (vo

l. %

)

Time (s)g

(2) ()

-1

Dh(AuNP):

9.5 nm Dh(CCMV):

28.3 nm

Dh(crystals):

1430 nm

Dh(amorphous):

~1200 nm

a b

0

0.2

0.4

0.6

0.8

1

0.1 10 1000 100000

No

rma

lise

d in

ten

sity [a

.u]

c

Inte

nsity (vo

l. %

)

Dh (nm)

0

1

0.1 100

g(2

) ()-

1

Time (s)

d

Dh(MF):

12.4 nm

Dh(amorphous):

~1600 nm

Dh(crystals):

1370 nm


S19

8. Agarose Gel Electrophoresis Mobility Shift Assay Data

Agarose gel EMSA was used as a complementary method to assess the formation of CCMV-AuNP

complexes. Formation of CCMV–AuNP complexes lowers the electrophoretic mobility of the free

virus and consequently a mobility shift towards the anode is observed. AuNPs were able to form

complexes with the CCMV (78 mg L-1

) as indicated by the mobility shift. Partial retardation of the

CCMV is observed between 0.02 and 0.3 AuNP/CCMV mass ratios, and the electrophoretic

mobility is completely blocked at higher AuNP concentrations.

Figure S14. Agarose gel images. Agarose gel electrophoresis of free CCMV and CCMV-AuNP complexes. Free

CCMV is on lane 1. Adding increasing amounts of the AuNPs disturbs the migration of the free virus due to complex

formation. The bands were first imaged using ethidium bromide fluorescence (upper image) and then with white light

(lower image).

9. Supplementary Information References

S1. Kanaras, A. G., Kamounah, F. S., Schaumburg, K., Kiely, C. J. & Brust, M. Thioalkylated tetraethylene glycol: a

new ligand for water soluble monolayer protected gold clusters. Chem. Commun. 2294-2295 (2002).

S2. Brust, M., Walker, M., Bethell, D., Schiffrin, D. J. & Whyman, R. Synthesis of thiol-derivatised gold

nanoparticles in a two-phase liquid-liquid system. Chem. Commun. 801-802 (1994).

S3. Miranda, O. R. et al. Enzyme-amplified array sensing of proteins in solution and in biofluids. J. Am. Chem. Soc.

132, 5285-5289 (2010).

S4. Haiss, W., Thanh, N. T. K., Aveyard & J. Fernig, D. G.Determination of size and concentration of gold

nanoparticles from UV-Vis spectra. Anal. Chem. 79, 4215-4221 (2007).

S5. Bancroft, J. B., Rees, M. W., Dawson, J. R. O., McLean, G. D. & Short, M. N. Some properties of a

temperature-sensitive mutant of cowpea chlorotic mottle virus. J. Gen. Virol. 16, 69-81 (1972).

S6. Verduin, B. J. M. The preparation of CCMV-protein in connection with its association into a spherical particle.

FEBS Lett. 45, 50-54 (1974).

cAuNP 0-160 (mg L-1)

mAuNP / mCCMV 0 0.02 0.04 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.2 1.6 2.0

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.lane

band* (lane n) / band* (lane 1)

(%)

100 94 85 56 32 12 7 1.5 - - - - - - - -

*


S20

S7. Cullity B.D., Stock S.R., Elements of X-Ray Diffraction, Prentice Hall (2001).

S8. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen

movements. J. Struct. Biol. 152, 36-51 (2005).

S9. Kostiainen, M. A. et al. Hierarchical self-assembly and optical disassembly for controlled switching of

magnetoferritin nanoparticle magnetism. ACS Nano 5, 6394-6402 (2011).

S10. Janner, A. Comparative architecture of octahedral protein cages. I. Indexed enclosing forms. Acta Cryst. A64,

494-502 (2008).

S11. Dolinsky, T. J., Nielsen, J. E., McCammon, J. A., Baker, N. A. PDB2PQR: an automated pipeline for the setup,

execution, and analysis of Poisson-Boltzmann electrostatics calculations. Nucl. Acids Res. 32, W665-W667.

(2004).

S12. Cheng, W. et al. Free-standing nanoparticle superlattice sheets controlled by DNA. Nat. Mater. 8, 519-525

(2009).

S13. Mikhaylov, G. et al. Ferri-liposomes as an MRI-visible drug-delivery system for targeting tumors and their

microenvironment. Nat. Nanotech. 6, 594-602 (2011).

S14. Uchida, M. et al. A human ferritin iron oxide nano-composite magnetic resonance contrast agent. Mag. Res. in

Med. 60, 1073-1081 (2008).

S15. Rohrer, M. et al. Comparison of magnetic properties of MRI contrast media solutions at different magnetic field

strengths. Invest. Radiol. 40, 715-724 (2005

S16. Berret, J.-F. et al. Controlled clustering of superparamagnetic nanoparticles using block copolymers: design of

new contrast agents for magnetic resonance imaging. J. Am. Chem. Soc. 128, 1755-1761 (2006).

S17. Laurent, S. et al. Magnetic iron oxide nanoparticles: synthesis, stabilization, vectorization, physicochemical

characterizations, and biological applications. Chem. Rev. 108, 2064-2110 (2008).

S18. Götz, J. & Zick, K. Local velocity and concentration of the single components in water/oil mixtures monitored

by means of MRI flow experiments in steady tube flow. Chem. Eng. Technol. 26, 59-68 (2003).

S19. Perez, J. M., Josephson, L., O'Loughlin, T., Hogemann, D. & Weissleder, R. Magnetic relaxation switches

capable of sensing molecular interactions. Nat. Biotech. 20, 816-820 (2002).

S20. Roch, A., Gossuin, Y., Muller, R. & Gillis, P. Superparamagnetic colloid suspensions: water magnetic relaxation

and clustering J. Magn. Magn. Mater. 293, 532-539 (2005).

S21. Mikhaylov, G. et al. Ferri-liposomes as an MRI-visible drug-delivery system for targeting tumours and their

microenvironment. Nat. Nanotech. 6, 594-602 (2011).


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