One significant barrier to the development of new generations of biocompatible materials, particularly tissue engineering hydrogels, is the difficulty of non-invasively evaluating the properties and performance of the biomaterial over time in vivo (1,2). Magnetic Resonance Imaging (MRI) is capable of whole-animal or -human imaging at high spatial and temporal resolution, and is an ideal modality for assessing hydrogel scaffolds in vivo (3). Although MRI possesses these advantages, it generally has low image contrast, making it difficult to distinguish the area of interest. Exogenous contrast agents (CAs) increase the relaxation rate (1/T1) of water protons and therefore improve image contrast. However, current clinically used CAs have low relaxivities and cannot be incorporated into biomaterials (4). Recently, researchers have been attaching small-molecule CAs to various macromolecules via either covalent or non-covalent interactions as a strategy to increase relaxivity (4). We have created a novel family of macromolecular CAs based on protein polymers to enable MRI tracking of biomaterials in vivo.

A family of protein polymer contrast agents was created by conjugating Gd(III) chelators onto evenly spaced lysines on recombinantly expressed protein polymers. Controlled cloning (5) and recombinant protein expression in E. coli were used to create the protein polymer backbone for the CAs. Inherent to this process, protein polymers are completely monodisperse and the amino acid sequence is exactly specified, allowing control over the density of reactive sites and length of the protein. Additionally, bioactive moieties, such as enyzymatic cleavage sites, could be included in the protein using controlled cloning. The chelators were conjugated onto the protein polymer in aqueous solution using peptide bond-forming agents (EDC and Sulfo-NHS). Due to the monodispersity of the protein polymers, the extent of conjugation can be characterized completely. We have demonstrated that we can modulate relaxivity through changing protein polymer length and lysine spacing. These CAs have been shown to be biodegradable by incubation with plasmin and virtually non-toxic to cells based on a viability assay. These protein polymer CAs have been crosslinked into tissue engineering hydrogels and shown dramatic contrast improvement. Since the enhanced contrast pixel volume due to the CAs can be tracked and should decrease as the material degrades, these CAs can be used to monitor in vivo scaffold degradation. In vivo experiments are in process to quantitatively analyze the degradation rate of protein polymer-based hydrogels.

References

1. Langer R., Tirrell D.A. Designing materials for biology and medicine. Nature, 428, 487, 2004.

2. Neves A.A., Medcalf N., Smith M., Brindle K.M. Evaluation of engineered meniscal cartilage constructs based on different scaffold geometries using magnetic resonance imaging and spectroscopy. Tissue Engr., 12, 53, 2006.

3. Bull S.R., Guler M.O., Bras R.E., Venkatasubramanian P.N., Stupp S.I., Meade T.J. Magnetic resonance imaging of self-assembled biomaterial scaffolds. Bioconjugate Chemistry, 16, 1343, 2005.

4. Artemov D., Bhujwalla Z., Bulte J. Magnetic resonance imaging of cell surface receptors using targeted contrast agents. Current Pharmaceutical Biotechnology, 5, 485, 2004.

5. Won J., Barron A. A new cloning method for the preparation of long repetitive polypeptides without a sequence requirement. Macromolecules, 35, 8281, 2002.