(28f) Formulation Design and Coacervation of a Recombinant Protein-Based Lung Sealant

After lung resections, sutures and staples are used to close lung tissue, but these techniques result in air leakages in ~50% of all cases [1]. Surgical sealants can supplement sutures and staples to prevent air leaks, but no available sealants are adhesive enough, elastic enough to allow normal lung expansion, and stable over time. We developed bioinspired protein-based sealants that combine adhesion from L-3,4-Dihydroxyphenalalanine (DOPA) residues native to mussel adhesive proteins with the mechanical properties of elastin. Our previous study demonstrated that these proteins are cytocompatible, provide the strongest bonds between substrates of any rationally designed protein when submerged in water, and can be easily applied underwater because they coacervate in physiological conditions [2]. In the current study, we examined the burst pressure adhesion, swelling properties, and elastic moduli of the adhesive formulations when crosslinked with iron nitrate, (hydroxymethyl)phosphonium chloride (THPC), or sodium periodate to tune the adhesive and mechanical properties of our protein adhesive. We also investigated the role of coacervation on the mechanical properties and adhesion of the glue formulations.

Proteins containing tyrosine (named ELP) were expressed in E. coli. Tyrosinase was used to convert the tyrosine in purified ELPs to adhesive DOPA (modified proteins named mELP). Burst pressure adhesion testing was based on ASTM standard F2392-04, and. Proteins were mixed with either iron, THPC, or periodate crosslinkers and applied to porcine intestine and cured in a humid chamber at 37°C. Burst pressures were compared to a commercially available topical sealant, New-Skin. Proteins were also crosslinked to form hydrogels for swelling and compression testing.

We evaluated the elastin-based proteins to determine whether their stiffness would match that of lung tissue. The ELP and mELP proteins that formed hydrogels were crosslinked with THPC and periodate (Figure 1A). The swollen gels had Young’s moduli (3-9 kPa) similar to that of lung tissue (1-5 kPa) [3]. We characterized the swelling behavior of the ELP and mELP crosslinked hydrogels over time (Figure 1B). Upon swelling, properties of a material can change, and adhesion can be reduced. Proteins crosslinked with THPC exhibited minimal volumetric shrinkage and equilibrated within one hour of application. mELP crosslinked with periodate swelled within the first 3 hours to over 3 times its application volume but equilibrated to a final volume less than the initial application volume within 72 hours of application. We measured the burst pressure strengths of our formulations with each different crosslinker (Figure 2). Upon application, all unmodified ELP formulations failed to create a seal across the substrate boundary due to dewetting of the surface. mELP + THPC also failed to form a seal likely due to the slower rate of network formation. mELP + iron attained an average burst pressure of 29.2 ± 13.6 kPa and mELP + periodate achieved burst pressures greater than 47 kPa. New-Skin, a commercially available topical sealant, had a burst pressure strength of 32.7 kPa ± 6.7 kPa. Thus, the two most promising formulations had adhesion strengths equal to or greater than a sealant meant only for topical applications.

The elastin-based DOPA-modified protein adhesives showed promise for biomedical applications including lung sealants. Our previous work demonstrated the ability of the material to set in wet environments and its cytocompatibility. In this study, we demonstrated the tunability of mechanical properties of the material through various crosslinking schemes to match the stiffness of soft tissues and quickly volumetrically equilibrate. Furthermore, we found both mELP + periodate and mELP + iron formulations to be promising for lung sealant application due to their high burst pressures.

References: [1] Mueller M.R. and Marzluf B.A., J Thorac Dis, 2014; 6: 14. [2] Brennan M.J. et al., Biomaterials, 2017; 124: 116-125. [3] Hinz B., Proc Am Thorac Soc, 2012; 9: 137-147.