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Multifunctional coatings combining bioactive peptides and affinity-based cytokine delivery for enhanced integration of degradable vascular grafts
Insufficient endothelialization of cardiovascular devices is a high-risk factor for implant failure. Presentation of extracellular matrix (ECM)-derived coatings is a well-known strategy to improve implant integration. However, the complexity of the system is challenging and strategies for applying multifunctionality are required. Here, we engineered mussel-derived surface-binding peptides equipped with integrin (c[RGDfK]) and proteoglycan binding sites (FHRRIKA) for enhanced endothelialization. Surface-binding properties of the platform containing l-3,4-dihydroxyphenylalanine (DOPA) residues were confirmed for hydrophilized polycaprolactone-co-lactide scaffolds as well as for glass and polystyrene. Further, heparin and the heparin-binding angiogenic factors VEGF, FGF-2 and CXCL12 were immobilized onto the peptide in a modular assembly. Presentation of bioactive peptides greatly enhanced human umbilical vein endothelial cell (HUVEC) adhesion and survival under static and fluidic conditions. In subsequent investigations, peptide-heparin-complexes loaded with CXCL12 or VEGF had an additional increasing effect on cell viability, differentiation and migration. Finally, hemocompatibility of the coatings was ensured. This study demonstrates that coatings combining adhesion peptides, glycosaminoglycans and modulators are a versatile tool to convey ECM-inspired multifunctionality to biomaterials and efficiently promote their integration. © 2020 The Royal Society of Chemistry.
Cell-free protein synthesis and in situ immobilization of deGFP-MatB in polymer microgels for malonate-to-malonyl CoA conversion
In the present work, microgels were utilized as a cell-free reaction environment to produce a functional malonyl-CoA synthetase (deGFP-MatB) under geometry-controlled transcription and translation. Our approach combines the straight-forward optimization of overall protein yield of an E. coli-based cell-free protein synthesis (CFPS) system based on concentration screening of magnesium and potassium glutamate, DNA as well as polyethylene glycol (PEG), and its innovative usage in microgel-based production of a key enzyme of the polyketide synthesis pathway. After partial modification of the carboxyl groups of hyaluronic acid (HA) with 5′-methylfuran groups via 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM)-activation, these were further functionalized with dibenzocyclooctyne (DBCO) and nitrilotriacetic acid (NTA) groups by bio-orthogonal [4+2] Diels-Alder cycloaddition to yield a bifunctional macromer. After coupling the DBCO groups with azide-functionalized DNA, containing the genetic information for deGFP-MatB, via strain-promoted azide-alkyne cycloaddition (SPAAC), the DNA-/NTA-functionalized HA macromer was utilized as base material together with maleimide-functionalized PEG (PEG-mal2) as the crosslinker to form bifunctional microgels utilizing water-in-oil (W/O) microemulsions. As-formed microgels were incubated with nickel sulfate to activate the NTA groups and provide binding sites for deGFP-MatB, which contained six histidine residues (His-tag) for that purpose. The optimized CFPS mixture was loaded into the microgels to initiate the formation of deGFP-MatB, which was detected by a clear increase in fluorescence exclusively inside the microgel volume. Functionality of both, the bound and the decoupled enzyme was proven by reaction with malonate to yield malonyl CoA, as confirmed by a colorimetric assay. © 2020 The Royal Society of Chemistry.