Nanofibrous Elastin-Like Protein As a Biomimetic Platform for Multifactorial Control of Cell-Matrix Interactions

Theme: Nanoparticles, Nanocomposites, and Nanoporous Materials for Bio-Applications

Title: Nanofibrous elastin-like protein as a biomimetic platform for multifactorial control of cell-matrix interactions

Authors: Patrick L. Benitez, Jeffery A. Sweet, and Sarah C. Heilshorn

Introduction: To produce physiologically relevant results, engineered extracellular matrices (eECMs) for in vitro studies of cell-matrix interactions must mimic the inherently nanoscale architecture, mechanics, and biochemistry of the native extracellular matrix (ECM). Given that cells sense the nanoscale articulation of each of these features,^{[1, 2]} ideal eECMs must offer not only a multifactorial design space but also a broadly mimetic microevironmental context. Here, to begin to meet this need, we electrospin a highly mimetic family of elastin-like proteins (ELPs) into a nanofibrous, stable, and broadly mimetic eECM. On the molecular level, ELPs have been engineered to mimic essential ECM proteins, combining a sequence derived from elastin, which confers native-like mechanical properties,^[3] with an extended sequence from fibronectin’s arginine-glycine-aspartic acid (RGD) ligand that enables specific adhesion.^[4] With limited self-assembly, though, ELPs require further processing to mimic the protein nanofibers that characterize the ECM: hence, electrospinning. To stabilize ELP nanofibers, we develop a crosslinking protocol that respects sequence specificity. Further characterization is done to demonstrate that relevant mechanics are achieved and specific signaling from the matrix is preserved during electrospinning and crosslinking.

Materials and Methods: ELPs, one containing the RGD ligand and an otherwise identical variant with the inert arginine-aspartic acid-glycine (RDG) sequence completely substituted for RGD, are recombinantly expressed and purified through inverse temperature cycling as previously described.^[4] Nanofibrous eECMs are electrospun from aqueous solutions of 35% w/w ELP, unless otherwise noted, at a flow rate of 200 µL h^-1, across a distance of 15 cm, and a voltage gap of 15 kV. Standard techniques are used to culture rodent marrow stromal cells (rMSCs) and to assess fiber morphology (scanning electron microscopy), fiber dissolution (bicinchoninic acid assay), mechanics (tensile testing), metabolism (resazurin), and actin/nuclear morphology of rMSCs (rhodamine-phalloidin, DAPI, confocal fluorescent microscopy, ImageJ). Fiber stabilization is achieved by crosslinking in two stages, vapor and aqueous. Briefly, eECMs are treated with glutaraldehyde vapor for 24 h followed by initial hydration in a minimal volume of 10x phosphate buffered saline. The initial hydration buffer was selected to decrease the kinetics of fiber disassembly while increasing the efficiency of crosslinking. After autoclaving and quenching reactive aldehydes in an excess of lysine, eECMs are washed and soaked in physiological buffer before use in further analyses.

Results and Discussion: Ribbon-like nanofibers (thickness: 190 nm, n=100) are electrospun from pure protein in aqueous solution. Nanofiber width (1.2 – 1.8 µm) is varied by modulating mass fraction of ELP in solution (20% – 30% w/w). This nanofibrous architecture is similar to structures seen in mammalian tissues such as the vascular tunica media.^[6] Two-stage crosslinking prevents disassembly of ELP nanofibers, as less than 2% of ELP mass dissolves over 2 weeks in physiological conditions (n=3). Mechanically, eECMs have properties (tensile modulus: 62 ± 6 kPa, n=5) similar to soft tissues.^[7] Increased rMSC survival in serum-free conditions on RGD-containing eECMs (p<0.002) demonstrates that the bioactivity of fibronectin-derived ligands is preserved during electrospinning and crosslinking. Similarly, a gradient of rMSC morphology is evident on eECMs with varying RGD content.

Conclusion: A native-like eECM is developed, and nanofibrous morphology, tensile mechanics, and specific bioactivity are characterized. In future work, we will broaden the compositional tunability of the eECM with respect to mechanics and nanoscale architecture, enabling highly biologically relevant, multifactorial studies of cell-matrix interactions.

Acknowledgements: We acknowledge funding from NSF DMR-0846363, NIH R21-AR062359, and NIH DP2-OD-006477 (S.C.H.).

References:

[1] Vogel V., Sheetz M., “Local force and geometry sensing regulate cell functions.” Nat. Rev. Cell Biol. 7: 265 (2006).

[2] Maheshwari G., Brown G., Lauffenburger D. A., Wells A., Griffith L. G., “Cell adhesion and motility depend on nanoscale RGD clustering.” J. Cell Sci. 113: 1677 (2000).

[3] Urry D. W., Hugel T., Seitz M., Gaub H. E., Sheiba L., Dea J., Xu J., Parker T. “Elastin: a representative ideal protein elastomer.” Phil. Trans. R. Soc. Lond. B 357: 169 (2002).

[4] Straley K. S., Heilshorn S. C., “Independent tuning of multiple biomaterial properties using protein engineering.” Soft Matter 5: 114 (2009).

[5] Pasqualini R., Koivunen E., Ruoslahti E., “A peptide isolated from phage display libraries is structural and functional mimic of an RGD-binding site on integrins.” J. Cell Biol. 130: 1189 (1995).

[6] Crissman R. S., Guilford W., “The three-dimensional architecture of the elastin-fiber network in canine hepatic portal system.” Am. J. Anat. 171: 401 (1984).

[7] Bergel, D. H., “The static elastic properties of the arterial wall.” J. Physiol. 156: 445 (1961).