Abstract Text: There is a major unmet need for non-invasive approaches to diagnosis, prognosis, and assessment of responses to treatment for neurodegenerative and neuroinflammatory conditions related to TBI. Molecular contrast brain MRI would allow safe, rapid, and quantitative visualization of many TBI-related conditions, and have multiple additional uses. We have designed a modular family of molecular contrast agents for brain MRI. Components include: 1) nanobodies binding to mouse transferrin receptor (mTfR) in brain capillary endothelia to promote receptor mediated transcytosis across the blood brain barrier (BBB), 2) additional nanobodies binding to specific molecular target of interest, 3) extremely small iron oxide nanoparticles (ESIONs) for MRI contrast, 4) Polyethylene glycol (PEG) coatings to prevent nanoparticle fouling in the blood and link ESIONs to nanobodies. The system is designed to be inherently modular; as new targets of interest are identified, target-specific nanobodies can be swapped out and replaced with new nanobodies without having to ‘start from scratch.’ We have generated nanobodies that bind to mTfR. The lead candidate called mTfRnb-M1 was generated by immunizing llamas with recombinant mTfR ectodomain. M1 is specific for its target. It does not appear to bind other proteins in mouse brain homogenates or serum. Furthermore, it does not interfere with holo-transferrin binding to mTfR. However, the affinity of mTfRnb-M1 is < 1 nM due to its slow off rate, which is not ideal for BBB transcytosis requiring unbinding from the receptor at the brain side of the BBB. We reasoned that since the endosomal compartments of brain endothelial cells are acidic, pH-dependent unbinding could enhance transcytosis. To confer pH dependent rapid unbinding, we created and screened multiple mTfRnb-M1 histidine mutants in and around the target binding site; histidine is protonated at acidic pH, which can change the structure of the protein and its interactions with targets. Several single, double, and triple histidine mutants conferred strongly pH dependent binding, as expected. We assessed the potential of the mTfR binding nanobodies to mediate BBB transcytosis in vivo by fusing them with neurotensin (NT). NT is a 13 amino acid peptide involved in hypothalamic-mediated thermoregulation, but it does not cross the BBB. Several M1-NT fusion constructs induced hypothermia in mice after iv injection. One mutant M1R56H, P96H, Y102H -NT produced >24-fold more potent hypothermic effects than the original M1 nanobody, indicating improved BBB crossing. Next, for proof-of-concept target binding in wild-type mice, we employed a nanobody called 13A7 which recognizes P2X7, an abundant ATP-gated ion channel in mouse brain. We constructed fusion proteins with mTfRnb-M1 mutants plus a tandem dimer of 13A7 and a 4th nanobody that binds albumin called Nb80. The albumin binding nanobody prolongs half-life in serum. This tetrameric construct, mTfRnb-M1R56H, P96H, Y102H-13A7-13A7-Nb80, when biotinylated and injected iv, crosses the BBB and binds in situ to targets in cortex and hippocampus. At 30-60 minutes, it primarily labels capillaries, whereas at 2, 4, and 8 hours post injection it strongly labels parenchymal cells. No signal was observed in brains of mice injected with negative control, biotinylated mTfRnb-M1AA-13A7-13A7-Nb80, which does not bind TfR or cross the BBB. In capillary depleted brain homogenates, the concentrations of the mTfRnb-M1P96H-13A7-13A7-Nb80 construct were as high as 3-5 nM. In parallel, we developed methods for assembling complete brain MRI molecular contrast agents and imaging them in vivo: 1) Thermal decomposition of iron oleate to form ESIONs. 2) Ligand exchange to replace oleate with phosphine oxide PEG2000-azide. 3) Click chemistry to link the azide to dibenzocyclooctyne-conjugated nanobody constructs. The complete brain MRI molecular contrast agents retained both the MRI T1 relaxation enhancing effects of the original ESIONs and the target binding properties of the nanobody constructs. As a proof-of-concept example, we obtained baseline MRI scans in living mice, injected known concentrations of ESION contrast agents into the striatum, and then rescanned the same mice. With non-linear co-registration, we obtained very high conspicuity of the injected ESIONs. Thus, in summary, we have made substantial progress in developing brain MRI molecular contrast agents. If successful, this line of inquiry will pave the way for a radical transformation in the way TBI-related neuroinflammation and neurodegeneration are assessed.