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
a
Electromagnetics Division, National Institute of Standards and Technology, Boulder, CO USA
Department of Chemistry and Biochemistry, University of Denver, CO USA
c
Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT USA
d
Department of Chemistry and Biochemistry, Indiana University, Bloomington, IN USA
b
Corresponding author:
Robert Usselman
Robert.usselman@gmail.com
1
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 proteinpolymer 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 P22Gd3+ cages [S2]. Previously, protein and Gd3+ concentrations for P22-Gd3+ (P22S39C-xAEMADTPA-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
2
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
3+
Gd Conc
Gd3+ Conc.
3+
a
Gd /cage
(mM)
Conc. (nM)
(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
a
Based on an interior volume of P22 with a radius of 29 nm.
b
In 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.
(μM)
Variable
Loading
(μM)
7200
Gd3+/cage
(μM)
10300
Gd3+/cage
(μM)
561
561
720
233
469
469
540
175
292
292
360
117
177
177
180
58
Gd(DTPA)2-
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+
3
concentrations as listed in Table S2 were vacuumed sealed under He (g) in Wildmad (WG-1364)
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
t


T1

M z (t )  M z , eq 1  be






(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.
4
The transverse relaxation times, T2, were calculated from Carr-Purcell-Meiboom-Gill (CPMG)
experiments with:
  Tt
M xy (t )  M xy (0) e 2






(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.
5
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.
6
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
7
Amplitude (arbitrary)
1120
1160
1200 1240
Field (mT)
1280
1320
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 ( ——).
8
Table S3. Average EPR linewidths at 80 and 150 K for samples with varying Gd3+
concentrations in the P22 cages.
Conc.
P22
of Gd
P22-Gd3+ EPR Line
Loading inside
Widths (mT)a
Gd/cage
P22
cages
a
b
(mM)
gx
gy
gz
1.7x103 b
28
27
18
7.0
2.9x103
46
28
23
6.0
4.6x103
74
34
21
6.3
1.0x104
189
36
29
7.2
Uncertainties in fitted linewidths are about 10%.
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.
9
Table S4. Power saturation of Gd3+ EPR signals at 80 K
sample
P1/2
2.0 mM Gd(DTPA)2-
Highest power
for linear
response
0.3 mW
1.0x104 Gd/P22 cage
12 mW
> 200 mW
2 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 Qband (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 twopulse 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.
10
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.
Concentration
T2e (s)
T1e (s)
0.1mM Gd3+
0.73
1.7, 0.70
0.2mM Gd3+
0.5mM Gd3+
1mM Gd3+
2mM Gd3+
5mM Gd3+
10mM Gd3+
20mM Gd3+
2mM Gd(DTPA)220mM Gd(DTPA)2-
0.69
0.58
0.50
0.43
0.31
0.21
0.09
0.50
0.2
1.5, 0.54
1.5, 0.6
1.4, 0.57
1.60, 0.67
1.4, 0.55
1.2, 0.35
0.96
1.6, 0.51
1.5, 0.52
References
S1.
S2.
S3.
L.O. Liepold, M.J. Abedin, E.D. Buckhouse, J.A. Frank, M.J. Young, T. Douglas:
Nano Lett 9, 4520-4526 (2009).
J. Lucon, S. Qazi, M. Uchida, G.J. Bedwell, B. LaFrance, P.E. Prevelige, T. Douglas:
Nature Chemistry 4, 781-788 (2012).
X.G. Lei, S. Jockusch, N.J. Turro, D.A. Tomalia, M.F. Ottaviani: Journal of Colloid
and Interface Science 322, 457-464 (2008).
11