Supporting the wound healing process by sending the appropriate cytokine signals can shorten healing time and overcome chronic inflammation syndromes. Even though adhesion peptides consisting of Arg‐Gly‐Asp (RGD) are commonly used to enhance cell‐surface interactions, peptide‐mediated cytokine delivery has not been widely exploited so far. Cytokines interact with high affinity with their cognitive receptors but also with sulfated glycosaminoglycans (GAGs), both of which form a base for incorporation of cytokines into functional biomaterials. Here, we report on a mussel‐derived surface coating as a prospective cytokine delivery system using covalently bound heparin‐mimetics, receptor derived chemokine binding peptides (RDP) and heparin binding peptides (HBP). The latter enabled non‐covalent immobilization of heparin on the surface followed by chemokine binding and release, while the former allowed direct non‐covalent chemokine immobilization The peptide displayed excellent binding to custom‐made polystyrene 96‐well plates, enabling convenient testing of several compounds. Released chemokine successfully induced migration in Jurkat cells especially for the non‐covalent heparin immobilization approach using HBPs as evaluated in a transwell assay. In comparison, heparin‐mimetic coatings, comprised of sulfated peptides and GAG derivatives, proved less efficient with respect to amount of immobilized chemokine and migratory response. Thus, our study provides a roadmap for further rational optimization and translation into clinics.

Biomaterials are designed to improve impaired healing of injured tissue. To accomplish better cell integration, we suggest to coat biomaterial surfaces with bio-functional proteins. Here, a mussel-derived surface-binding peptide is used and coupled to CXCL12 (stromal cell-derived factor 1α), a chemokine that activates CXCR4 and consequently recruits tissue-specific stem and progenitor cells. CXCL12 variants with either non-releasable or protease-mediated-release properties were designed and compared. Whereas CXCL12 was stabilized at the N-terminus for protease resistance, a C-terminal linker was designed that allowed for specific cleavage-mediated release by matrix metalloproteinase 9 and 2, since both enzymes are frequently found in wound fluid. These surface adhesive CXCL12 derivatives were produced by expressed protein ligation. Functionality of the modified chemokines was assessed by inositol phosphate accumulation and cell migration assays. Increased migration of keratinocytes and primary mesenchymal stem cells was demonstrated. Immobilization and release were studied for bioresorbable PCL-co-LC scaffolds, and accelerated wound closure was demonstrated in an ex vivo wound healing assay on porcine skin grafts. After 24 h, a significantly improved CXCL12-specific growth stimulation of the epithelial tips was already observed. The presented data display a successful application of protein-coated biomaterials for skin regeneration.

Vanadium-dependent haloperoxidases (VHPOs) are biotechnologically valuable and operationally versatile biocatalysts that do not require complex electron shuttling systems. These enzymes share remarkable active-site structural similarities yet display broadly variable reactivity and selectivity. The factors dictating substrate and halogen specificity and, thus, a general understanding of VHPO reaction control still need to be discovered. This work's strategic single-point mutation in the cyanobacterial bromoperoxidase AmVHPO facilitates a selectivity switch to allow aryl chlorination. This mutation induces loop formation absent in the wild-type enzyme, and that interacts with the neighboring protein monomer, creating a tunnel to the active sites. Structural analysis of the substrate-R425S-mutant complex reveals a substrate-binding site at the interface of two adjacent units. There, residues Glu139 and Phe401 interact with arenes, extending the substrate residence time close to the vanadate cofactor and stabilizing intermediates. Our findings validate the long-debated existence of direct substrate binding and provide detailed VHPO mechanistic understanding. This work will thus pave the way for a broader application of VHPOs in diverse chemical processes.