A rechargeable anti-thrombotic coating for blood-contacting devices.

Despite the potential of anti-thrombogenic coatings, including heparinized surfaces, to improve the performance of blood-contacting devices, the inevitable deterioration of bioactivity remains an important factor in device failure and related thrombotic complications. As a consequence, the ability to restore the bioactivity of a surface coating after implantation of a blood-contacting device provides a potentially important strategy to enhance its clinical performance. Here, we report the regeneration of a multicomponent anti-thrombogenic coating through use of an evolved sortase A to mediate reversible transpeptidation. Both recombinant thrombomodulin and a chemoenzymatically synthesized ultra-low molecular weight heparin were repeatedly and selectively immobilized or removed in a sequential, alternating, or simultaneous manner. The generation of activated protein C (aPC) and inhibition of activated factor X (FXa) was consistent with the molecular composition of the surface. The fabrication of a rechargeable anti-thrombogenic surface was demonstrated on an expanded polytetrafluoroethylene (ePTFE) vascular graft with reconstitution of the surface bound coating 4 weeks after in vivo implantation in a rat model.

In situ regeneration of bioactive coatings enabled by an evolved Staphylococcus aureus sortase A.

Surface immobilization of bioactive molecules is a central paradigm in the design of implantable devices and biosensors with improved clinical performance capabilities. However, in vivo degradation or denaturation of surface constituents often limits the long-term performance of bioactive films. Here we demonstrate the capacity to repeatedly regenerate a covalently immobilized monomolecular thin film of bioactive molecules through a two-step stripping and recharging cycle. Reversible transpeptidation by a laboratory evolved Staphylococcus aureus sortase A (eSrtA) enabled the rapid immobilization of an anti-thrombogenic film in the presence of whole blood and permitted multiple cycles of film regeneration in vitro that preserved its biological activity. Moreover, eSrtA transpeptidation facilitated surface re-engineering of medical devices in situ after in vivo implantation through removal and restoration film constituents. These studies establish a rapid, orthogonal and reversible biochemical scheme to regenerate selective molecular constituents with the potential to extend the lifetime of bioactive films.

Reprogramming the specificity of sortase enzymes.

Staphylococcus aureus sortase A catalyzes the transpeptidation of an LPXTG peptide acceptor and a glycine-linked peptide donor and has proven to be a powerful tool for site-specific protein modification. The substrate specificity of sortase A is stringent, limiting its broader utility. Here we report the laboratory evolution of two orthogonal sortase A variants that recognize each of two altered substrates, LAXTG and LPXSG, with high activity and specificity. Following nine rounds of yeast display screening integrated with negative selection, the evolved sortases exhibit specificity changes of up to 51,000-fold, relative to the starting sortase without substantial loss of catalytic activity, and with up to 24-fold specificity for their target substrates, relative to their next most active peptide substrate. The specificities of these altered sortases are sufficiently orthogonal to enable the simultaneous conjugation of multiple peptide substrates to their respective targets in a single solution. We demonstrated the utility of these evolved sortases by using them to effect the site-specific modification of endogenous fetuin A in human plasma, the synthesis of tandem fluorophore-protein-PEG conjugates for two therapeutically relevant fibroblast growth factor proteins (FGF1 and FGF2), and the orthogonal conjugation of fluorescent peptides onto surfaces.

Antifouling glycocalyx-mimetic peptoids.

The glycocalyx of the cell is composed of highly hydrated saccharidic groups conjugated to protein and lipid cores. Although components of the glycocalyx are important in cell-cell interactions and other specific biological recognition events, a fundamental role of the glycocalyx is the inhibition of nonspecific interactions at the cell surface. Inspired by glycoproteins present in the glycocalyx, we describe a new class of synthetic antifouling polymer composed of saccharide containing N-substituted polypeptide (glycopeptoid). Grafting of glycopeptoids to a solid surface resulted in a biomimetic shielding layer that dramatically reduced nonspecific protein, fibroblast, and bacterial cell attachment. All-atom molecular dynamics simulation of grafted glycopeptoids revealed an aqueous interface enriched in highly hydrated saccharide residues. In comparison to saccharide-free peptoids, the interfacial saccharide residues of glycopeptoids formed a higher number of hydrogen bonds with water molecules. Moreover, these hydrogen bonds displayed a longer persistence time, which we believe contributed to fouling resistance by impeding interactions with biomolecules. Our findings suggest that the fouling resistance of glycopeptoids can be explained by the presence of both a 'water barrier' effect associated with the hydrated saccharide residues as well as steric hindrance from the polymer backbone.

Electrospun catechol-modified poly(ethyleneglycol) nanofibrous mesh for anti-fouling properties.

Electrospun nanofibrous mesh composed of catechol-conjugated 8-arm PEG (8cPEGa) and thiolated PLGA (PLGA-SH) was prepared with various blending ratios of PLGA-SH and 8cPEGa. Cross-linking between the two polymers via catechol-thiol reactions and catechol-catechol conjugation was performed by brief soaking with sodium periodate solution. The chemical conjugation of PLGA-SH and 8cPEGa in the nanofibrous mesh was confirmed by the spectral differences of the Raman spectra and changes in the thermal properties. The crosslinked meshes showed lower degradation rates and their fibrous morphologies remained intact even after 15 days. When the blend ratio of 8cPEGa was increased from 0 to 50%, the crosslinked meshes showed a dramatic decrease in the water-contact angles due to the surface-exposed PEG chains tethered on the mesh. The crosslinked meshes had superior anti-fouling effect on protein and mammalian cell binding in proportion to the amount of 8cPEGa in the mesh compared to non-crosslinked meshes.

Oligonucleotide chip for the diagnosis of HNF-1 alpha mutations.

Mutations in HNF-1 alpha cause maturity-onset diabetes of the young (MODY) type 3, which is the most prevalent MODY subtype in most countries. In the present study, we investigated an oligonucleotide microchip for the detection of the known HNF-1 alpha mutations. We first optimized the coupling chemistries for covalent immobilization of allele-specific oligonucleotides on aldehyde (CHO)- and thiocyanate (NCS)-activated glass slides and compared their hybridization efficiencies. CHO-glass was found to provide a more favorable environment for hybridization than NCS-glass, whereas the binding capacity of NCS-glass for amine-activated oligonucleotide was much greater than with CHO-glass. We also investigated the effects of the length of the capture probes on the hybridized signals. To determine the presence of HNF-1 alpha mutations in a human sample, we prepared an oligonucleotide chip from selected mutation sites of exon2 from HNF-1 alpha. Cy3-labeled RNA target probes were obtained by in vitro transcription of promoter-tagged PCR products from a wild-type blood sample and subsequent fragmentation. Hybridization of the chip with the RNA target probes successfully identified all of the genotypes for the tested sites. This work demonstrates that oligonucleotide chip-based analysis is a good candidate for routine clinical testing for HNF-1 alpha mutations.