PEGylation of lysine residues improves the proteolytic stability of fibronectin while retaining biological activity.

Excessive proteolysis of fibronectin (FN) impairs tissue repair in chronic wounds. Since FN is essential in wound healing, our goal is to improve its proteolytic stability and at the same time preserve its biological activity. We have previously shown that reduced FN conjugated with polyethylene glycol (PEG) at cysteine residues is more proteolytically stable than native FN. Cysteine-PEGylated FN supported cell adhesion and migration to the same extent as native FN. However, unlike native FN, cysteine-PEGylated FN was not assembled into an extracellular matrix (ECM) when immobilized. Here, we present an alternative approach in which FN is preferentially PEGylated at lysine residues using different molecular weight PEGs. We show that lysine PEGylation does not perturb FN secondary structure. PEG molecular weight, from 2 to 10 kDa, positively correlates with FN-PEG proteolytic stability. Cell adhesion, cell spreading, and gelatin binding decrease with increasing molecular weight of PEG. The 2-kDa FN-PEG conjugate shows comparable cell adhesion to native FN and binds gelatin. Moreover, immobilized FN-PEG is assembled into ECM fibrils. In summary, lysine PEGylation of FN can be used to stabilize FN against proteolytic degradation with minimal perturbation to FN structure and retained biological activity.