Novel multimodal molecular imaging of Vitamin H (Biotin) transporter activity in the murine placenta

Vitamin H (biotin) is delivered to the fetus transplacentally by an active biotin-transport mechanism and is critical for fetal development. Our objective was to develop a comprehensive MRI technique for mapping biotin transporter activity in the murine placenta. Visualization of transporter activity can employ MRI’s unique T2*-dependent signal ‘off-switch’, which is triggered by transporter mediated aggregation of biotinylated contrast agent (b-BSA-Gd-DTPA). MRI data were collected from pregnant mice after administration of b-BSA-Gd-DTPA and analyzed using a new sub-voxel biophysical signal model. Validation experiments included competition with native biotin, comparative tests using PET, histology, and ICPMS. MRI signal was governed by binding, aggregation, and clearance of biotin (confirmed by histology). Signal dynamics reflected the placenta’s perfusion pattern modulated by biotin transporter activity and trophoblast mediated retention, and were in congruence with a three-compartment sub-voxel model. Pre-saturation of the transporters with free biotin suppressed b-BSA-Gd-DTPA uptake. The results were confirmed by PET, histology and ICPMS. The presented MRI-based platform allows to track activity of essential molecular transporters in the placenta, reflecting a transporter-mediated uptake, followed by retention and aggregation, and recycling associated with the large b-BSA-Gd-DTPA conjugate. The presented DCE-MRI technique can furthermore be used to map and characterize microstructural compartmentation and transporter activity without exposing the fetus to contrast media.


Contrast preparation and administration
Biotin-BSA-GdDTPA [19] was synthesized from bovine serum albumin (BSA) by conjugation with biotin and GdDTPA (biotin-BSA-GdDTPA; approximately 80 kDa; r1 relaxivity of 177 mM −1 s −1 per albumin and 7.55 mM −1 s −1 per Gd at 4.7 T). During dynamic contrast enhanced (DCE) MRI experiments, an intravenous bolus dose of biotin-BSA-GdDTPA (10 mg/mouse (Gd: 92 μmol/kg) in 200 μl of PBS) was injected via a tail-vein catheter. The biotin label enabled the distribution of the contrast material to be visualized in histological sections.

DCE-MRI Imaging and analysis
MRI experiments were performed on a 9.4 tesla BioSpec Magnet 94/20 USR system (Bruker, Germany) equipped with a gradient-coil system capable of producing pulsed gradients of up to 40 gauss/cm in each of the three directions. A quadrature volume coil, with 72-mm inner diameter and a homogeneous RF field of 100 mm along the axis of the magnetic field, was used as both transmitter and receiver. During MRI scanning, mice were anesthetized with isoflurane (3% for induction, 1-2% for maintenance) in O2 (1 liter/min) delivered through a nasal mask. Once anesthetized, the animals were secured in a head-holder and were imaged in a supine position. Respiration rate was monitored and maintained at 30-45 breaths/min, while body temperature was maintained at 37 o C via circulating warm water.

Post-mortem histology, immunohistochemistry, and fluorescence microscopy
Following MRI imaging, pregnant animals were euthanized with an overdose of pentobarbital, uteri were exposed, and embryos were counted, weighed, and numbered according to their position along the uterine horn.
Placentae were fixed in Carnoy mixture, embedded in paraffin, and sectioned serially at 4-μm thickness. Sections were deparaffinized with a xylene substitute (Safeclear II; Fisher Scientific Company LLC, Kalmazoo, MI) for 5 min; rehydrated sequentially with 100%, 95%, and 70% ethanol and double-distilled water for 5 min each, and then equilibrated in PBS for 5 min. Slides were then incubated with Citrate buffer (PH=6) for one hour in a pressure cooker for antigen retrieval. After nonspecific binding was blocked using a solution of 20% normal horse serum and 0.2% of Triton-X in PBS for 1.5 h at room temperature, sections were incubated in blocking solutions containing 2% normal horse serum and 0.2% Triton-X Anti-Bovine Albumin Antibody (1:100, Bio-Rad, Hercules, After drying, compound #2 (5 mg) was dissolved into 300 μL of anhydrous dimethyl sulfoxide, and added to the vial and heated at 135 o C for 30 min. After the synthesis, the vial was cooled to room temperature and diluted into 4 mL of water. The crude reaction solution was pushed through and trapped onto a C18 cartridge, first prepared with 3 mL of ethanol and 10 mL of water. The trapped product was washed with another 10 mL of water to remove any unreacted [ 18 F]F -. The final product, (3) was then eluted off of the C18 Sep-Pak with 1 mL of ethanol, concentrated down to dryness and reconstituted into 25 μL of ethanol for dose preparation described below. Radiopurity of the final compound was checked with radio-HPLC and was found to be greater than 98%.
It should be mentioned that while the synthesis of the radiolabeling precursor, #2 (see Fig. S6), was accomplished in one step, the precursor was not stable due to a high reactivity. To ensure good radiolabeling yields, the precursor was therefore synthesized no more than one week before the study, and kept under vacuum until use, to ensure a dry environment.
Pregnant CD1 mice (E18-20) were anesthetized with 2-3% isoflurane in oxygen, maintained at normal body temperature, with respiratory monitoring. Animals were first injected with 3.5 mg/g of body weight Omnipaque, an iodine-based CT contrast agent, followed by the prepared Biotin PET tracer described above, also via tail vein injection.
Animals were placed on the scanner bed and dynamic PET data were acquired for 20 min, followed by a 5-minute CT scan (80kVp), concluding with a 40-minute dynamic PET scan.
Immediately following the conclusion of the 2 nd PET acquisitions, the mouse was sacrificed immediately for biodistribution. Organs and tissues of interest, including each fetus, associated placenta and uterus, were harvested, weighed and the radioactivity was measured using a gamma counter. Data were decay corrected to time of animal sacrifice and calculated as the percent injected dose per gram of tissue (%ID/g).
CT images were reconstructed using a Modified Feldkamp Algorithm. The PET images were reconstructed using a 3D-OSEM (Ordered Subset Expectation Maximization) algorithm (24 subsets and 3 iterations), with random, attenuation, and decay correction. Final images were processed using Vivoquant.

Statistical analysis of uptake of PET tracer in placentae and fetuses:
The uptake, %ID/g, of [ 18 F]Biotin in the fetuses and placentae in each group were compared by one-way ANOVA followed by a Tukey's post-hoc test. There were 39 fetuses and placentae for the non-blocking studies and 37 fetuses and placentae for the blocking studies.      * p < 0.05 ** p < 0.01 *** p < 0.001 **** p < 0.0001