Supplementary Information to Accompany

Gadolinium-loaded Viral Capsids as Magnetic Resonance Imaging Contrast Agents*

Robert J. Usselman,†a Shefah Qazi,c Priyanka Aggarwal,b Sandra S. Eaton,b Gareth R. Eaton,b

Trevor Douglas,d and Stephen E. Russeka

aElectromagnetics Division, National Institute of Standards and Technology, Boulder, CO USAbDepartment of Chemistry and Biochemistry, University of Denver, CO USAcDepartment of Chemistry and Biochemistry, Montana State University, Bozeman, MT USAdDepartment of Chemistry and Biochemistry, Indiana University, Bloomington, IN USA

Corresponding author:Robert UsselmanRobert.usselman@gmail.com

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Experimental Section

Preparation of samples.

All materials were analytical grade, purchased from either Sigma-Aldrich or Fisher

Scientific, and used as received unless otherwise noted. All water was deionized with a Millipore

NANOpure water purification system. The DTPA-NCS was purchased from Macrocyclics

(Dallas, TX). Gd-DTPA-NCS was made according to published procedures [S1]. Briefly, 7.41

mg (11.40 µmoles) DTPA-NCS was dissolved in 101.3 µL of 900 mM sodium bicarbonate and

827 µL water. Once the DTPA-NCS was completely dissolved, 11.4 µL GdCl3 (900 mM in water,

10.26 µmoles) was added to the solution and stirred for 3 hrs at room temperature. The solution

was subsequently diluted to 10 mM with DMSO (200 µL) and added to the protein-polymer

conjugate P22S39C-xAEMA [2], in specific subunit mole ratios.

The following describes reaction of Gd-DTPA-NCS with P22S39C-xAEMA using 100:1

ratio of Gd to subunit, which was found to give 7200 Gd/cage (17 Gd/subunit). 425 µL (4.25

µmoles) Gd-DTPA-NCS (10 mM in DMSO/H2O) was added dropwise to 1.0 mL (0.0425 µmoles

subunit, 2 mg/mL carbonate buffer, pH 9.0) P22S39C-xAEMA, while vortexing the protein

solution. The mixture was allowed to sit overnight at 4 ºC followed by purification to remove

excess Gd-DTPA-NCS by pelleting and resuspending the protein twice. All protein samples that

have been chemically modified were analyzed via UV-VIS (UV-Vis; Model 8453, Agilent, Santa

Clara, CA), dynamic light-scattering (DLS; 90Plus particle-size analyzer, Brookhaven

Instrument, Holtsville, NY) and SDS-PAGE on 10-20 % gradient Tris-glycine gels (Lonza).

Protein was detected by staining with Coomassie blue. Certain commercial equipment,

instruments, or materials are identified in this document. Such identification does not imply

recommendation or endorsement by the National Institute of Standards and Technology, nor

does it imply that the products identified are necessarily the best available for the purpose.

Protein purification, sample analysis, and determination of protein and gadolinium

concentrations were previously described and the samples were consistent with polymeric P22-

Gd3+ cages [S2]. Previously, protein and Gd3+ concentrations for P22-Gd3+ (P22S39C-xAEMA-

DTPA-Gd) samples with different loading factors were analyzed by inductively coupled plasma

mass spectrometry ICP-MS [S2]. From the ICP-MS results, a standard curve was made for Gd3+

concentration and NMR T1 measurements at 2.1 T.  Total Gd3+ concentration was determined

from the NMR standard curve. Protein capsid concentration was determined by subtracting the

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Gd(DTPA)2- absorbance at 280 nm from the total Abs280. The uncertainty in concentration for

the 10300 Gd3+/cage was about 12% based on standard curves derived from ICP-MS, NMR, and

UV-Vis. Similar uncertainties were expected for the other samples based on previous

measurements [S2].  

Table S1. P22 Cage Gd3+ loading factors, Gd3+ concentrations inside P22 cages, P22 cage concentrations and global Gd3+ concentrations.

P22 Loading Local P22 P22 Cage Global

Gd3+/cageGd3+ Conc

(mM)a Conc. (nM)Gd3+ Conc.

(μM)1730 28 102 177b

2870 46 102 292b

4590 74 102 469b

5500 89 102 561b

7200 117 100 720c

10300 189 22.6 233c

aBased on an interior volume of P22 with a radius of 29 nm. bIn solution for NMRD.c Stock solution from which solutions for NMRD were prepared.

Table S2. Global Gd3+ concentrations in solutions for the four NMRD experiments.

Gd(DTPA)2- Variable Loading

7200 Gd3+/cage

10300 Gd3+/cage

(μM) (μM) (μM) (μM)561 561 720 233469 469 540 175292 292 360 117177 177 180 58

NMR Relaxivity measurements.

A vertical broadband NMR spectrometer system was used to measure proton relaxation

times by an inversion recovery sequence with repetition times of 30 s for T1 relaxation times and

CPMG (Carr-Purcell-Meiboom-Gill) sequence for T2 relaxation times, respectively. Relaxation

times were measured at NMR frequencies that ranged from 20 MHz to 300 MHz (0.49 to 7.0 T)

at 294 K. To minimize radiation damping, 60 µL of the buffered P22-Gd3+ samples with Gd3+

concentrations as listed in Table S2 were vacuumed sealed under He (g) in Wildmad (WG-1364)

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capillary tubes. Relaxivity r1 and r2 at each magnetic field was calculated as the slope of plots of

1/T1 or 1/T2 vs. Gd3+ concentration, respectively.

Values of T1 were calculated as

M z ( t )=M z , eq(1−be

− tT 1)

(S1)

Mz,eq(t) nuclear spin magnetization on the z axis at time t in units of seconds

Mz(∞ ) equilibrium state of the nuclear spin magnetization in the z axis

T1 the time constant for the recovery of the z component of the nuclear spin

Magnetism

b fitting parameter between 1 and 2 that reflects variable extent of inversion

Figure S1. Example of NMR inversion recovery data at 0.5 T for P22-Gd3+ variable loading sample with 5500 Gd3+/cage, cage concentration of 102 nM to give a global Gd3+ concentration of 561 μM.

The transverse relaxation times, T2, were calculated from Carr-Purcell-Meiboom-Gill (CPMG)

experiments with:

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M xy ( t )=M xy( 0)(e− t

T2 )(S2)

Mxy(t) the transverse magnetization in the xy plane at time t in units of seconds

Mxy(0) initial transverse magnetization at time zero

T2 the decay constant for the component of the magnetization M

perpendicular to applied magnetic field

Figure S2. Example of NMR Carr-Purcell-Meiboom-Gill (CPMG) data at 0.5 T for P22-Gd3+ variable loading sample with 5500 Gd3+/cage, cage concentration of 102 nM to give a global Gd3+ concentration of 561 μM.

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Figure S3. Example of NMR 1/T1 relaxation rates at 0.5 T for the three types of P22-Gd3+ samples and Gd(DTPA)2-. The slopes of the linear fits give r1 values.

Figure S4. Example of NMR 1/T2 relaxation rates at 0.5 T for the three types of P22-Gd3+ samples and Gd(DTPA)2-. The slopes of the linear fits give r2 values.

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Continuous Wave Q-band EPR Spectra at 80 and 150 K

To characterize the dipolar spin-spin interaction in the P22-Gd3+ samples, EPR

spectroscopy was used to measure the linewidths as a function of Gd3+ loading. Spectra were

recorded at 80 or 150 K on a Bruker E580 spectrometer at 34 GHz (Q-band). P22-Gd3+ samples

for EPR were prepared by adding an equal volume of glycerol to the carbonate buffer to ensure

glass formation rather than crystallization as the sample was quickly cooled. Samples were

transferred into quartz capillaries and centrifuged for 4 min at 1000 X g. Because of zero-field

splitting (ZFS), the EPR spectra extend over hundreds of mT. The most prominent features of

these spectra are the ms=1/2 transitions that are observed near g ~2. These spectral segments

are shown in Fig. S5. Signals were simulated using locally-written software with anisotropic g

values: gx = 1.964, gy = 1.994, and gz=1.995. Within estimated uncertainties, linewidths are the

same at 80 and 150 K so average values are shown in Table S3. The linewidths probably have

contributions from distributions in g values and in ZFS as well as dipolar interactions. The

concentration dependent contribution reflects the increase in dipolar interactions [S3]. Within

estimated uncertainties of the fitting parameters the line widths along the principal axes increase

significantly with increasing concentration, consistent with substantial dipolar interactions at

these high local concentrations. The increases are larger along gx and gy than along gz. For the

samples with 1.0x104 Gd3+/cage the linewidths along each of the axes are about 1 to 8 mT larger

than for 1.7x103 Gd3+/cage, which is a contribution to T2e of about 1x10-10 to 9x10-10 s at 80 K.

This contribution to T2e is very large relative to typical T2e for low concentrations of Gd3+ of

about 5x10-7 s at 80 K (Table S5). The strong dipolar interactions are consistent with the

expectations that the Gd3+ is concentrated in the interior of the P22 particle [S2] and may

contribute to the larger values of r1 and r2 for the sample with the higher Gd3+ loading of the

cages

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Figure S5. Q-band EPR spectra of the ms= 1/2 transitions for P22-Gd3+ with 4.6x103 Gd3+/cage ( _ _ _ ) or 1.0x104 Gd3+/cage ( ——).

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Table S3. Average EPR linewidths at 80 and 150 K for samples with varying Gd3+

concentrations in the P22 cages.

P22 Loading Gd/cage

Conc. of Gd inside P22

cages

P22-Gd3+ EPR Line Widths (mT)a

(mM) gx gy gz

1.7x103 b 28 27 18 7.0

2.9x103 46 28 23 6.0

4.6x103 74 34 21 6.31.0x104 189 36 29 7.2

a Uncertainties in fitted linewidths are about 10%.b Linewidth determined only at 150 K.

Power saturation at 80 K

To characterize the changes in electron spin relaxation due to Gd3+-Gd3+ interactions

inside the P22 cages, the amplitudes of the Gd3+ EPR signals in the g~ 2 region were recorded at

80 K as a function of microwave power for 2.0 mM Gd(DTPA)2- in pH 9 carbonate

buffer:glycerol (1:1) and for P22-Gd3+ with 10300 Gd3+/cage. These experiments were

performed at X-band (9.7 GHz) because resonator tuning is more reproducible than at the 34

GHz. Data were plotted as a function of P to obtain a power saturation curve. For the two

samples, the highest powers at which the signal increases linearly with P are shown in Table

S4. P1/2 , the power at which the signal amplitude is half of the value predicted in the absence of

saturation, also is listed. These measurements indicate that electron spin relaxation at 80 K for

the Gd3+ in the P22 cages is dramatically enhanced compared with that for Gd(DTPA)2- at 2.0

mM. Although the -NCS substituent on the DTPA and attachment to the polymeric framework

impacts the local environment such that the ZFS for Gd3+ in the P22-DTPA samples is different

than for Gd(DTPA)2-, the differences in ZFS are likely to cause much smaller changes in

relaxation than are caused by the changes in concentration.

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Table S4. Power saturation of Gd3+ EPR signals at 80 K

sample Highest power for linear response

P1/2

2.0 mM Gd(DTPA)2- 0.3 mW 2 mW

1.0x104 Gd/P22 cage 12 mW > 200 mW

Pulsed EPR measurement of Gd3+ electron spin relaxation times at 80 K

Pulsed EPR experiments were performed at 80K on a Bruker E-580 spectrometer at Q-

band (34 GHz). Samples of Gd(DTPA)2- in 1:1 water:glycerol and of P22-Gd3+ in 1:1

buffer:glycerol were examined. Electron spin-spin relaxation times (T2e) were measured by two-

pulse spin echo using pulse length of 40 and 80 ns. The initial time for data acquisition was 200

ns, which is limited by the resonator ring-down. Electron spin-lattice relaxation times (T1e) were

measured by inversion recovery using 80ns-40ns-80ns pulses. The relaxation times T2e and T1e

were obtained by fitting single or double exponentials to the data using Bruker E-580 software.

The fit to the inversion recovery curves was substantially better for the sum of two exponentials

than for a single exponential, which may reflect overlapping contributions from transitions with

different values of ms.

For the P22-Gd3+ samples the spin echoes were weak, and attributed to small amounts of

Gd3+ that were not inside the cages. The relaxation times from Gd3+ inside the cages were too

short to measure by pulsed EPR. Values of T1e and T2e for aquo Gd3+ and Gd(DTPA)2- are

summarized in Table S5. Relaxation times are weakly concentration dependent in the range of

0.1 to 2 mM, but become more strongly dependent at higher concentrations.

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Table S5. Concentration dependence of T1e and T2e for Gd3+ and Gd(DTPA)2- at 80 K and 34 GHz in 1:1 water:glycerol.

References

S1. L.O. Liepold, M.J. Abedin, E.D. Buckhouse, J.A. Frank, M.J. Young, T. Douglas: Nano Lett 9, 4520-4526 (2009).

S2. J. Lucon, S. Qazi, M. Uchida, G.J. Bedwell, B. LaFrance, P.E. Prevelige, T. Douglas: Nature Chemistry 4, 781-788 (2012).

S3. X.G. Lei, S. Jockusch, N.J. Turro, D.A. Tomalia, M.F. Ottaviani: Journal of Colloid and Interface Science 322, 457-464 (2008).

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Concentration T2e (s) T1e (s)

0.1mM Gd3+ 0.73 1.7, 0.70

0.2mM Gd3+ 0.69 1.5, 0.540.5mM Gd3+ 0.58 1.5, 0.61mM Gd3+ 0.50 1.4, 0.572mM Gd3+ 0.43 1.60, 0.675mM Gd3+ 0.31 1.4, 0.5510mM Gd3+ 0.21 1.2, 0.3520mM Gd3+ 0.09 0.962mM Gd(DTPA)2- 0.50 1.6, 0.5120mM Gd(DTPA)2- 0.2 1.5, 0.52