Genetically encoded biocompatible anti-coagulant protein-coated coronary artery stents drive endothelialization.

In response to the critical demand for advancements in coronary artery stents, this study addresses the challenges associated with arterial recoil and restenosis post-angioplasty and the imperative to encourage rapid re-endothelialization for minimizing thrombosis risks. We employed an innovative approach inspired by mussel adhesion, incorporating placental anticoagulant protein (AnnexinV) on stent design. The introduction of a post-translationally modified catecholic amino acid L-3,4-dihydroxyphenylalanine (L-Dopa), mimicking mussel characteristics, allowed for effective surface modification of Stainless steel stents through genetic code engineering in AnnexinV (AnxDopa). The efficacy of AnxDopa was analyzed through microscale thermophoresis and flow cytometry, confirming AnxDopa's exceptional binding with phosphatidylserine and activated platelets. AnxDopa coated stainless steel demonstrates remarkable bio-, hemo-, and immuno-compatibility, preventing smooth muscle cell proliferation, platelet adhesion, and fibrin formation. It acts as an interface between the stent and biological fluid, which facilitates the anticoagulation and rapid endothelialization. Surface modification of SS verified through XPS analysis and contact angle measurement attests to the efficacy of AnxDopa mediated surface modification. The hydrophilic nature of the AnxDopa-coated surface enhanced the endothelialization through increased protein absorption. This approach represents a significant stride in developing coronary stents with improved biocompatibility and reduced restenosis risks, offering valuable contributions to scientific and clinical realms alike.

Collagen Mimicry with a Short Collagen Model Peptide.

Mimicking triple helix and fibrillar network of collagen through collagen model peptide(CMP) with short GPO tripeptide repeats is a great challenge. Herein, a minimalistic CMP comprising only five GPO repeats [(GPO)5 ] is presented. This novel approach involves the fusion of ultrashort peptide with the synergetic power of π-system and ß-sheet formation to short CMP (GPO)5 . Accordingly, a hydrogel-forming, fluorenylmethoxycarbonyl (Fmoc)-functionalized ultrashort peptide (NFGAIL) is fused at the N-terminus and phenylalanine at the C-terminus of (GPO)5 (Fmoc-NFGAIL-(GPO)5 -F-COOH, FmP-5GPO). At room temperature, it forms a robust triple helix in aqueous buffer solution and has a relatively high melting point of 35 °C. The fluorenyl motif stabilizes the triple helix by aromatic π-π interactions as in its absence, triple helix is not formed. NFGAIL, which forms a ß-sheet, also aids in triple helix stabilization via intermolecular hydrogen bonding and hydrophobic interactions. FmP-5GPO forms highly entangled nanofibrils with a micrometer length, which have excellent cell viability. The achievement of stable triple helix and fibrils in such a short CMP(FmP-5GPO) sequence is a challenging feat, and its significance in CMP-based biomaterials is undeniable. The present strategy highlights the potential for developing new CMP sequences through intelligent tuning of fusion peptides and GPO repeats.

Three-dimensional tailor-made collagen-like proteins hydrogel for tissue engineering applications.

Despite the potential tunable properties of blank slate collagen-like proteins (CLP), an alternative to animal-originated collagen, assembling them into a stable 3D hydrogel to mimic extracellular matrix is a challenge. To address this constraint, the CLP (without hydroxyproline, CLPpro) and its variants encoding functional unnatural amino acids such as hydroxyproline (CLPhyp) and 3,4-dihydroxyphenylalanine (CLPdopa) were generated through genetic code engineering for 3D hydrogel development. The CLPhyp and CLPdopa were chosen to enhance the intermolecular hydrogen bond interaction through additional hydroxyl moiety and thereby facilitate the self-assembly into a fibrillar network of the hydrogel. Hydrogelation was induced through genipin as a cross-linker, enabling intermolecular cross-linking to form a hydrogel. Spectroscopic and rheological analyses confirmed that CLPpro and its variants maintained native triple-helical structure, which is necessary for its function, and viscoelastic nature of the hydrogels, respectively. Unlike CLPpro, the varients (CLPhyp and CLPdopa) increased pore size formation in the hydrogel scaffold, facilitating 3T3 fibroblast cell interactions. DSC analysis indicated that the stability of the hydrogels got increased upon the genetic incorporation of hydroxyproline (CLPhyp) and dopa (CLPdopa) in CLPpro. In addition, CLPdopa hydrogel was found to be relatively stable against collagenase enzyme compared to CLPpro and CLPhyp. It is the first report on 3D biocompatible hydrogel preparation by tailoring CLP sequence with non-natural amino acids. These next-generation tunable CLP hydrogels open a new venue to design synthetic protein-based biocompatible 3D biomaterials for tissue engineering applications.

Growth factor-mimicking 3,4-dihydroxyphenylalanine-encoded bioartificial extracellular matrix like protein promotes wound closure and angiogenesis.

The present work reports a new route to prepare a "smart biomaterial" by mimicking long-acting cellular growth factor showing enhanced cell-material interactions by promoting cell proliferation and angiogenesis. For that, reactive non-proteogenic amino acid 3,4-dihydroxyphenylalanine (DOPA) was genetically introduced into an intrinsic triple-helical hierarchical structure forming protein to initiate hierarchical self-assembly to form a macromolecular structure. The self-assembled scaffold displayed vascular endothelial growth factor mimicking the pro-angiogenic reactive group for repairing and remodeling of damaged tissue cells. We customized the recombinant collagen-like protein (CLP) with DOPA to promote rapid wound healing and cell migrations. Selective incorporation of catechol in variable and C-terminal region of CLP enhanced interaction between inter- and intra-triple-helical collagen molecules that resulted in a structure resembling higher-order native collagen fibril. Turbidity analysis indicated that the triple-helical CLP self-assembled at neutral pH via a catechol intra-crosslinking mechanism. After self-assembly, only DOPA-encoded CLP formed branched filamentous structures suggesting that catechol mediated network coordination. The catechol-encoded CLP also acted as a "smart material" by mimicking long-acting cellular growth factor showing enhanced cell-material interactions by promoting cell proliferation and angiogenesis. It eliminates release rate, stability, and shelf-life of hybrid growth factor conjugated biomaterials. The newly synthesized CLP has the potential to promote accelerated cell migration, pro-angiogenesis, and biocompatibility and could be used in the field of implantable medical devices and tissue engineering.