Use of a Genetically Engineered Protein for the Design of a Multivalent MRI Contrast Agent

Abstract

The majority of clinically used contrast agents (CAs) for magnetic resonance imaging have low relaxivities and thus require high concentrations for signal enhancement. Research has turned to multivalent, macromolecular CAs to increase CA efficiency. However, previously developed macromolecular CAs do not provide high relaxivities, have limited biocompatibility, and/or do not have a structure that is readily modifiable to tailor to particular applications. We report a new family of multivalent, biomacromolecular, genetically engineered protein polymer-based CAs; the protein backbone contains evenly spaced lysines that are derivatized with gadolinium (Gd(III)) chelators. The protein's length and repeating amino acid sequence are genetically specified. We reproducibly obtained conjugates with an average of 8 -9 Gd(III) chelators per protein. These multivalent CAs reproducibly provide a high relaxivity of 7.3 mM -1 s -1 per Gd(III) and 62.6 mM -1 s -1 per molecule. Furthermore, they can be incorporated into biomaterial hydrogels via chemical crosslinking of remaining free lysines, and provide a dramatic contrast enhancement. Thus, these protein polymer CAs could be a useful tool for following the evolution of tissue engineering scaffolds.One significant barrier to the development of new generations of biocompatible materials, particularly tissue engineering hydrogels, is an inability to non-invasively evaluate the properties and performance of the biomaterial over time (1-6). Magnetic Resonance Imaging (MRI) is capable of whole animal or human imaging at high spatial and temporal resolution, and is an ideal modality for evaluating tissue engineering scaffolds in vivo (7-11). Exogenous contrast agents (CAs) increase the relaxation rate (1/T 1 ) of water protons and therefore improve image contrast. However, current clinically used CAs have low relaxivities (3-7 mM -1 s -1 ) (12) and thus must be used at high concentrations for useful MRI signal enhancement (12).T 1 CAs provide positive contrast by employing a paramagnetic ion (typically gadolinium, Gd (III)). The efficacy of a contrast agent is dominated by three parameters: q, the number of We have designed, synthesized and characterized a macromolecular T 1 contrast agent based on artificial proteins. The backbone of these multivalent MRI contrast agents is derived from E. coli expression of monodisperse protein polymers. Gd(III) chelators were chemically conjugated to the backbone to create contrast agents that display high relaxivity in solution and when incorporated into a hydrogel. The covalent incorporation of these protein polymer contrast agents into a gel allows the potential for imaging tissue engineering scaffolds. Here, we report the design, synthesis, and characterization of a novel family of multivalent, macromolecular CAs based on genetically engineered proteins with repetitive sequences that form the backbone for subsequent chemical modification with Gd(III) chelators. These "protein polymer" CAs have high relaxivities in aqueous solution. Mo...

An Extracellular MRI Polymeric Contrast Agent That Degrades at Physiological pH

“…These ‘small fragments’ do not tumble slower than the small molecule contrast agent. Our polymer relaxivity measurements are similar to other polymeric contrast agents 2-5 and peptide-based contrast agents 31, 32 .…”

Synthesis and Characterization of a New Class of Cationic Protein Polymers for Multivalent Display and Biomaterial Applications

“…Recently, using the same conjugation chemistry, our lab has reported the coupling of gadolinium chelators to these protein polymer scaffolds for use as MRI contrast agents. 49 …”

“…For example, PEGylation of the scaffold resulted in higher solubility than unconjugated protein polymer (results not shown). We hypothesize that given the flexibility of the backbone and the initially random coil structure of the protein polymer, the conformation is unlikely to change significantly with the addition of a few pendant groups, depending on the structure of the conjugated moieties (preliminary data with Gd conjugated 49 protein polymers). Furthermore, we hypothesize that the low efficiency of grafting in the conjugates described here should lead to only minor changes in solution properties.…”

“…Gels crosslinked with glutaraldehyde showed increased MRI contrast over controls. 49 In the present study, we incorporated K8 30 -AF488 into the protein polymer hydrogel by chemical crosslinking with BS 3 . As expected, the fluorescence of AF488 was not affected by the incorporation of the protein polymer scaffold into a chemically crosslinked hydrogel (Figure 9C).…”

Real-time, non-invasive monitoring of hydrogel degradation using LiYF4:Yb3+/Tm3+ NIR-to-NIR upconverting nanoparticles

“…MRI is attractive because of its high spatial resolution, but it requires magnetically active materials as contrast agents and sophisticated instrumentation. 8 Fluorescence-based assays therefore remain the most widely used method for hydrogel tracking in vivo. For example, recently, Artzi et al fluorescently labeled polyethylene glycol (PEG) hydrogels to track their degradation in vivo and developed a correlation with in vitro degradation.…”