Relaxivity of 3 nm IONPs at 3.0 T, 4.7 T, and 9.4 T
3 nm IONPs were synthesized following the methods of Kim et al. and validated using dynamic light scattering and electron microscopy 42. Synthesis and ligand exchange were performed at the Chemistry and Synthesis Center (CSC) in NIH. Iron chloride hexahydrate (FeCl3·6H2O), 80% oleic acid, Polyethylene glycol methyl ether (PEG750), tetrahydrofuran (THF) and dichloromethane were purchased from Sigma Aldrich. Sodium oleate was purchased from Tokyo Chemical Industry (Portland, Oregon, USA). Hydroxyl-PEG-Azide 2k (OH-PEG-N3) was obtained from Biochempeg. Iron concentrations were measured by inductively coupled plasma optical emission spectroscopy (ICP-OES) method using Agilent 5900 SVDV ICP-OES (Santa Clara, California, USA). A series of phantoms were made from 3 nm IONPs suspended in 1x PBS and diluted to 0.02, 0.04, 0.06, 0.08, and 0.1 mM [Fe]. 1xPBS served as a control. Both longitudinal and transverse relaxation rates of 3 nm IONP were assessed at 3.0 T, 4.7 T, and 9.4 T magnetic field.
The longitudinal relaxation rate constant (R1, 1/T1, s− 1) of 3 nm IONP was assessed using modified fast inversion recovery (MFIR)43. The inversion MR data was collected after passing the null point to suppress macromolecule effects on R1 estimation 43,44. The repetition time (TR) was 5.0 s for 3.0 T and 8.0 s for both 4.7 T and 9.4 T where the echo time (TE) was 3.0 ms for all magnetic field strength. The inversion delay time points were optimized for each magnetic field as follows; 1) 3.0 T -13 inversion delay time points at 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 3000, 3500, 4000, and 4500 ms. 2) 4.7 T – 17 inversion delay time points at 800, 900, 1000, 1100, 1200, 1300, 1600, 1900, 2300, 2600, 2900, 3300, 3600, 3900, 5500, 6500, 7900 ms. 3) 9.4 T – 15 inversion delay time points at, 1100, 1200, 1300, 1600, 1900, 2300, 2600, 2900, 3300, 3600, 3900, 5000, 6000, 7000, 7900 ms. The R1 was estimated using Bayesian analysis toolbox (http://bayesiananalysis.wustl.edu/). The longitudinal relaxivity, r1 (mM− 1s− 1), of 3 nm IONP was estimated from the R1 of 3 nm IONP at five different iron concentration, 0.02 to 0.1 mM [Fe]. The obtained r1 (mM− 1s− 1) was used to calculate the theoretical R1 enhancement by r1 (mM− 1s− 1) ∈ [Fe] (mM).
The transverse relaxation rate constant (R2, s− 1, 1/T2) of 3 nm IONPs was assessed using multi echo train MR sequence, 24 ms echo spacing and 20 echo train at both 3.0 T and 9.4 T. The TR was 5.0 s for 3.0 T and 8.0 s for 9.4 T. The R2 was estimated using Bayesian analysis toolbox (http://bayesiananalysis.wustl.edu/). Similar to r1, the transverse relaxivity, r2 (mM− 1s− 1), of 3 nm IONP was estimated from the R2 of IONP at five different iron concentration, 0.02 to 0.1 mM [Fe].
In vivo subjects
Human brain MRI images were performed under the National Institutes of Health Institutional Review Board (NIH IRB) approved protocol (NCT00001711). Informed consent was obtained prior to a health subject R1 mapping MR scans, which were conducted in the Clinical Center at the National Institutes of Health (NIH). Human brain MRI image sets for R2 mapping were acquired from a health subject following informed consent at the National Intrepid Center of Excellence. All human related procedures and methods were performed in accordance with the relevant guidelines and regulations.
All animal experiments were conducted under protocols approved by the National Institute of Neurological Disorders and Stroke (NINDS)/ National Institute on Deafness and Other Communication Disorders (NIDCD) Animal Care and Use Committee in the NIH Clinical Center. All in vivo animal related procedures and methods were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the NIH and were in accordance with ARRIVE guidelines (https://arriveguidelines.org). C57BL6 female mice at 10 weeks of age were purchased from Jackson labs and used at 12-weeks of age. A total 11 mice were used in this study, five mice at 4.7 T and six mice at 9.4 T. Among the 6 mice scanned at 9.4 T, five mice, (mouse ID 06–10), were from our previous report41.
In vivo MRI of mouse brain at 4.7 T and 9.4 T
In vivo mouse brain MR scans were performed on Bruker 4.7 T and 9.4 T scanner (Bruker, Ettlingen, Germany) with imaging gradient 180 mT/m for 4.7 T and 260 mT/m for 9.4 T on Paravision 6.01 platform. All employed naïve mice underwent two independent MR scans, MP2RAGE or multi echo T2 scan, within one-week.
At 4.7 T, a single channel RF coil was used to acquire MP2RAGE data at 160 µm ×160 µm × 480 µm voxel size, zero-filled to 80 µm ×80 µm ×80 µm, covering the entire brain with 8 averages and 60 minutes total scan time. The thick image slice was selected to increase signal to noise ratio at magnetic field, 4.7 T. The MP2RAGE imaging parameters were TR/TE/TI1/TI2 (ms) = 8000/3/1100/2600, 9-degree flip angle, and 640 ms segment duration with 2 inversion for a k-space plane. In vivo mouse brain R2 map at 4.7 T was not pursued due to the limitation of gradient maximum rise time (or slew rate).
At 9.4 T, 4-channel RF coil used to acquire MP2RAGE data at 160 µm ×160 µm × 160 µm voxel size, zero-filled to 80 µm ×80 µm ×80 µm, covering the entire brain with 6 averages and 95 minutes total scan time. The MP2RAGE imaging parameters were TR/TE/TI1/TI2 (ms) = 8000/3/1300/3600, 9-degree flip angle, and 640 ms segment duration with 2 inversion for a k-space plane. The MR protocol of MP2RAGE at 9.4 T is the same as our previous report41. The T1 maps of in vivo mouse brain was estimated from raw MP2RAGE and converted into R1 maps. In addition, multi echo T2 weighted images were acquired at 160 µm ×160 µm × 240 µm voxel size, zero-filled to 80 µm ×80 µm ×80 µm, covering the entire brain with TR/ echo spacing/ echo train number/ average/ total scan time = 8.0 s/ 8.0 ms/ 20/ 1/ 10 minutes. The R2 map was estimated using the Bayesian analysis toolbox (http://bayesiananalysis.wustl.edu/). The mouse brain T1 (1/R1) was 1.88 s in the current study. Thus, the short TR might cause T1 contamination on mouse brain R2 map. The R2 value of in vivo mouse brain at 9.4 T dependence on TR was examined by comparing 2D MR sequence having long TR (8.0 s) with 3D having short TR (1.0 s), see Supplementary Fig. 4.
In vivo MRI of human brain at 3.0 T
Two different 3.0 T human scanners were used for in vivo human brain MR scan with two normal adult subjects. In vivo human brain MP2RAGE MR scans were performed on a Siemens Prisma 3.0 T magnet (Erlangen, Germany) with 80 mT/m imaging gradient and 32 channel radio frequency (RF) coil. The first normal adult subject underwent two independent 3-dimensional MP2RAGE scans within one-week, at the same time of the day on each session. The MP2RAGE images were acquired with the following parameters: echo time (TE) = 2.88 ms, repetition time (TR) = 5000 ms, inversion delay times TI1/TI2 = 700/2500 ms, field of view (FOV) = 256×256×192 mm, matrix = 256×256×192, flip angles = 4-degree for TI1 and 5-degree for TI2, band width = 240 hz/pixel, GRAPPA acceleration factor = 3, total scan time = 8 minutes 56 second. The obtained MP2RAGE data had 1.0 ×1.0 ×1.0 mm isotropic voxel size. The T1 maps of in vivo human brain were estimated from raw MP2RAGE data using the vendor providing imaging handling software, Syngo MR D13D. The obtained T1 maps were converted into R1 maps (= 1/T1).
In vivo human brain T2 mapping scan using T2 weighted imaging (T2WI) sequence were performed on a GE SIGNA 3.0 T magnet (Waukesah, Milwaukee, USA) with 80 mT/m imaging gradient and 32 channel radio frequency (RF) coil. The second normal adult subject underwent two independent T2 mapping MR scans within 2 hours. The second subject took a short break between the 1st and 2nd MR scan. 2-dimensional T2WI were acquired with the following parameters: TR = 4200 ms, FOV = 240 mm x 240 mm, matrix = 256 x 256, slice thickness = 1.2 mm, total number of slices = 142, TE = 30 ms and 110 ms, total scan time = 12 minutes 32 seconds. T2 map was calculated using linear T2 MR signal decay approach with two echo time (TE) MR signals following the methods from a previous report45. The 30 ms echo time was selected as short echo time T2WI to avoid myelin water effect in T2 map calculation46 and resulted in improved reproducibility (Supplementary Fig. 7).. The 110 ms echo time was selected as long echo time T2WI ensuring about 50 % signal to noise ratio of 30 ms echo time T2WI. The resulting T2 map was converted into R2 maps (= 1/T2).
Data and image processing
For both 4.7 T and 9.4 T, two MP2RAGE data were acquired from the same mouse within one-week time frame producing two independent R1 maps. Using the advanced normalization tools (ANTs, http://stnava.github.io/ANTs/)47, the 2nd R1 map was co-registered to the 1st R1 map. After co-registration the absolute ΔR1 (|ΔR1|) map between 1st R1 map and the 2nd R1 map co-registered to 1st R1 map was calculated, producing a baseline. The hypothesized R1 enhancing effects on in vivo mouse brain induced by 3 nm IONPs were simulated by digitally adding R1 values to the 2nd R1 map co-registered to 1st R1 map. This approach simulates the result that could hypothetically be obtained if a subject were scanned at baseline, administered an IONP contrast agent, and then scanned again at an appropriate time. The hypothetical R1 enhancement of the 3 nm IONPs in mouse brain was calculated from equation [1].
ΔR1 = r1 (mM− 1s− 1)× [Fe] (mM) [1]
The digital simulated additional R1 values were randomly placed in both small (from 1 to 8 voxels) and larger (from 20 to 30 voxels) zones in the cortex of the 2nd R1 map co-registered to 1st R1 map. It was assumed that all brain tissues have the same degree of R1 enhancing effect from the same concentrations of 3 nm IONP. This procedure was performed for five hypothetical IONP iron concentrations: 0.02, 0.04, 0.06, 0.08, and 0.1 mM. Similar to the baseline |ΔR1| map, the |ΔR1| maps between the 1st R1 map and the co-registered 2nd R1 map having the hypothesized effects of 3 nm IONP were calculated to assess the sensitivity of MP2RAGE derived R1 maps to 3 nm IONP contrast agents. In vivo mouse brain R2 maps underwent the same procedures where the hypothetical R2 enhancement of the 3 nm IONPs in mouse brain was calculated from equation [2].
ΔR2 = r2 (mM− 1s− 1) × [Fe] (mM) [2]
The same procedures were performed on in vivo human brain R1 maps obtained at 3.0 T. Following image co-registration, the baseline |ΔR1| was obtained. The digital simulated additional R1 values, which were calculated from the r1 of the 3 nm IONPs at 3.0 T, were randomly placed in both small (from 1 to 2 voxels) and larger (from 3 to 9 voxels) zones in representative human brain regions. The |ΔR1| map between the 1st R1 map and co-registered 2nd R1 map after digitally added R1 equivalent to several concentrations of 3 nm IONP were calculated. Analogous procedures were conducted using R2 maps.