DEVELOPMENT OF A CALMODULIN-BASED BIOMATERIAL FOR IN SITU DRUG DELIVERY
There is growing interest in harnessing genetically engineered polymers to develop responsive biomaterials. Unlike their synthetic counterparts, genetically engineered polymers are produced without the use of toxic reagents, and they can easily be programmed to incorporate desirable hydrogel properties, including bioactivity, biodegradability, and monodispersity. We have recently developed a very promising copolymeric hydrogel that is based on the calcium-dependent protein, calmodulin (CaM)1. For our system, CaM and M13, a CaM-binding peptide, were incorporated into genetically engineered polymers with intervening cleavable sequences. Spectroscopic and multiple particle tracking (MPT) studies demonstrated that these polymers self-assemble through calcium-stabilized, noncovalent crosslinking to form a soft viscoelastic material. MPT further revealed that gelation is concentration-dependent. Collagenase digests show that the protein polymers are, indeed, selectively degraded through specific cleavage by matrix metalloproteinases. The modularity, specific, high affinity, calcium dependent interaction between these protein copolymers and the stimuli-responsiveness of this system suggest its potential as a flexible scaffold for biomedical applications.
We can tune both the protein matrix properties and covalently modify the matrix with specific biological or chemical agents that together allow very specific tailoring of applications. To that end, we are pursuing a collaborative project with Prof. Davita Watkins in our home department focused on covalent modification of the polymers for in situ drug delivery. The concept is to modify polymers with NSAIDs prior to assembly and then to assembly in vivo drug depot at the site of inflammation. This method allows delivery of common anti-inflammatory drugs at high levels locally to avoid the pleiotropic toxic effects from systemic delivery.
Further, a critical feature of our copolymers is that they are completely soluble in water from storage as a sterile dry powder indicating that they can be reconstituted as needed, a critical attribute that allows their use for situations that may emerge in the isolated environment encountered by astronauts in extended missions. Experiments described herein are directed toward the use of our hydrogel scaffold for in situ drug delivery and wound healing, including antibiotic delivery2, 3and scaffolding of tissue regrowth4.
Optimization of polymer design features to diversify applications. To build on our successful initial studies, we are very interested in exploring how changes in the composition of the polymeric components will affect the hydrogel properties. To that end,we are undertaking a redesign of the M13-based polymer to increase the number of repeats and to improve its solubility.
Testing the effect of Ionic Liquids on Polymer solubility and function. Redesigned PCLP3 polymers will be assayed for solubility in aqueous solutions of Ionic Liquids and Organic co-solvents. The effect of suitable solubilizing agents on intermolecular interactions with the CaM and CCLP3 will be studied.
Covalent modification of CaM-based polymer for in situ drug delivery. We are collaborating with the Watkins lab in the Dept of Chemistry and Biochemistry at UM to covalently modify the CaM-based polymer with model compounds for the purpose of developing the linkage chemistry in our system.
Assessment of biocompatibility of hydrogel. In order to move these studies forward, we must address the important issue of biocompatibility of the hydrogel. To this end we will assess whether our hydrogel is toxic to mammalian cells through collaboration with Dr. Shabana Khan in the National Center for Natural Products Research at the University of Mississippi.
 Fox, C. S., Berry, H. A., and Pedigo, S. (2020) Development and Characterization of Calmodulin-Based Copolymeric Hydrogels, Biomacromolecules 21, 2073-2086.
 Batoni, G., Maisetta, G., and Esin, S. (2016) Antimicrobial peptides and their interaction with biofilms of medically relevant bacteria, Biochim Biophys Acta 1858, 1044-1060.
 Bayramov, D. F., and Neff, J. A. (2016) Beyond conventional antibiotics -New directions for combination products to combat biofilm, Advanced drug delivery reviews.
 Seidi, A., Ramalingam, M., Elloumi-Hannachi, I., Ostrovidov, S., and Khademhosseini, A. (2011) Gradient biomaterials for soft-to-hard interface tissue engineering, Acta Biomater. 7, 1441-1451.
Pedigo, S. and Shea, M. A. “Quantatitive Endo GluC Footprinting of Cooperative Ca2+-binding to Calmodulin: Proteolytic Susceptibility of E31 and E87 Indicates Inter-domain Interactions” (1994) Biochemistry 34: 1179-1196.
Pedigo, S. and Shea, M. A. “Discontinuous Equilibrium Titrations of Cooperative Calcium Binding to Calmodulin Monitored by 1D 1H-Nuclear Magnetic Resonance Spectroscopy” (1995) Biochemistry 34: 10676-10689.
Shea, M. A., Verhoeven, A. S. and Pedigo, S. “Calcium Induced Domain Interactions of Calmodulin Revealed by Quantitative Thrombin Footprinting of Arg37 and Arg106” (1996) Biochemistry 35: 2943-2957.
Shea, M. A., Sorensen, B. R., Pedigo, S., Verhoeven, A. S. “Proteolytic footprinting titrations for estimating ligand binding constants and detecting pathways of conformational switching in calmodulin” (2000) Methods in Enzymology 323: 254-301.
Hobson, K. F., Housley, N. A. and Pedigo, S. “Ligand-linked stability of mutants of the C-domain of calmodulin” (2005) Biophysical Chemistry 114(1): 43-52.
Fox, C.S., Berry, H.A. and Pedigo, S. “Development and Characterization of Calmodulin-Based Copolymeric Hydrogels” (2020) Biomacromolecules 21:2073-2086.