Abstract Body:

Manganese is a popular agent as a non-lanthanide metal for MRI contrast [1]. Due to its high uptake in mitochondria-rich organs, its use as a contrast agent is especially popular in the liver, pancreas and kidneys. Manganese-based contrast agents also heavily rely on its chelation chemistry where its stability with a chelate is required to provide control over its release of free ions – critical to its imaging function and for potential safety concerns [1]. Despite the popularity of manganese for paramagnetic contrast, its chelator agents, such as porphyrins and polycarboxylic acids, have relied mainly on the ability to non-covalently bind manganese to increase its relaxivity by slower rotation. These chelation chemistries offer little in terms of designable parameters to tune this relaxivity or maintain its signal for the duration of imaging. On the other hand, a protein-based MRI agent offers a tunable amino acid sequence library to control chelation via avidity to a paramagnetic metal, such as gadolinium or manganese. This modularity can allow for improvements in relaxivity, reduced toxicity, and for scaffolding other engineered moieties such as protein binders for targeting. In particular, manganese is a common endogenous cofactor for many proteins such as arginase or prolidase [2, 3]. In fact, naturally derived proteins such the metalloregulatory protein MntR or divalent metal transporter 1 (DMT1) [4, 5] have been assessed as protein-based chelators capable of providing robust Mn-based signal and have shown the ability to be subcellularly imaged when fused to a targeting protein [6]. Furthermore, sustained release of molecular targets have also been shown to be well controlled using hydrogel-based biomaterials that allow for slow diffusion from the matrix [7]. We have recently developed a series of protein-based, upper critical solution temperature (UCST) hydrogels which are computationally controlled for varying material strengths and gelation kinetics [8, 9]. Using a best performer from this set, Q5, capable of fast gelation, high material strength, and high gel-sol transition temperature, we explore its ability to bind manganese for localized imaging. Manganese-bound Q5 transitions into a stable hydrogel (Figure 1a) capable of binding manganese within its matrix as well as binding specifically to the supramolecularly assembled protein fibers (Figure 1b-d). We show that the hybrid Q5-manganese hydrogels possess T1-brightening expected of manganese alone using a custom Teflon well-plate setup. This allows for optimization of manganese concentration in our setup (Figure 1g) which we use for Q5 dosage of manganese in its characterization (Figure 1h).  We further show that manganese-bound Q5 hydrogels are also capable of providing prolonged stability for imaging in vivo through intraarticular injection as compared to a manganese or buffer control which shows limited signal through rapid circulation. The hybrid protein hydrogel-manganese system here introduces a new and uniquely modular platform for localized imaging.

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