Multifunctional and biologically active matrices from multicomponent polymeric solutions

The present invention relates to a biologically active functionalized electrospun matrix to permit immobilization and long-term delivery of biologically active agents. In particular the invention relates to a functionalized polymer matrix comprising a matrix polymer, a compatibilizing polymer and a biomolecule or other small functioning molecule. In certain aspects the electrospun polymer fibers comprise at least one biologically active molecule functionalized with low molecular weight heparin. Examples of active molecules that may be used with the multicomponent polymer of the invention include, for example, a drug, a biopolymer, for example a growth factor, a protein, a peptide, a nucleotide, a polysaccharide, a biological macromolecule or the like. The invention is further directed to the formation of functionalized crosslinked matrices, such as hydrogels, that include at least one functionalized compatibilizing polymer capable of assembly

Transient dynamic mechanical properties of resilin-based elastomeric hydrogels

The outstanding high-frequency properties of emerging resilin-like polypeptides (RLPs) have motivated their development for vocal fold tissue regeneration and other applications. Recombinant RLP hydrogels show efficient gelation, tunable mechanical properties, and display excellent extensibility, but little has been reported about their transient mechanical properties. In this manuscript, we describe the transient mechanical behavior of new RLP hydrogels investigated via both sinusoidal oscillatory shear deformation and uniaxial tensile testing. Oscillatory stress relaxation and creep experiments confirm that RLP-based hydrogels display significantly reduced stress relaxation and improved strain recovery compared to PEG-based control hydrogels. Uniaxial tensile testing confirms the negligible hysteresis, reversible elasticity and superior resilience (up to 98%) of hydrated RLP hydrogels, with Young's modulus values that compare favorably with those previously reported for resilin and that mimic the tensile properties of the vocal fold ligament at low strain (<15%). These studies expand our understanding of the properties of these RLP materials under a variety of conditions, and confirm the unique applicability, for mechanically demanding tissue engineering applications, of a range of RLP hydrogels

Conformational and aggregation properties of a pegylated alanine-rich polypeptide

The conformational and aggregation behavior of PEG conjugates of an alanine-rich polypeptide (PEG-c17H6) were investigated and compared to that of the polypeptide equipped with a deca-histidine tag (17H6). These polypeptides serve as simple and stimuli-responsive models for the aggregation behavior of helix-rich proteins, as our previous studies have shown that the helical 17H6 self-associates at acidic pH and converts to β-sheet structures at elevated temperature under acidic conditions. In the work here, we show that PEG-c17H6 also adopts a helical structure at ambient/subambient temperatures, at both neutral and acidic pH. The thermal denaturation behavior of 17H6 and PEG-c17H6 is similar at neutral pH, where the alanine-rich domain has no self-association tendency. At acidic pH and elevated temperature, however, PEGylation slows β-sheet formation of c17H6, and reduces the apparent cooperativity of thermally induced unfolding. Transmission electron microscopy of PEG-c17H6 conjugates incubated at elevated temperatures showed fibrils with widths of ∼20-30 nm, wider than those observed for fibrils of 17H6. These results suggest that PEGylation reduces β-sheet aggregation in these polypeptides by interfering, only after unfolding of the native helical structure, with interprotein conformational changes needed to form β-sheet aggregates.National Center for Research Resources (NCRR); Center for Neutron Science (U.S. Dept. of Commerce

Multifunctional and biologically active matrices from multicomponent polymeric solutions

A functionalized electrospun matrix for the controlled-release of biologically active agents, such as growth factors, is presented. The functionalized matrix comprises a matrix polymer, a compatibilizing polymer and a biomolecule or other small functioning molecule. In certain aspects the electrospun polymer fibers comprise at least one biologically active molecule functionalized with low molecular weight heparin

Development of a collagen-based scaffold for sequential delivery of antimicrobial agents and pdgf genes to chronic wounds

Chronic wounds are a global health burden affecting more than 5 million people in the United States alone. The complex wound microenvironment causes variable therapeutic outcomes following treatment with commercially available products. Wound infection is one of the major barriers in healing of wounds and localized delivery of antimicrobials is necessary for treatment. Furthermore, growth factors play a vital role in orchestrating the wound healing process through enhancement of cell proliferation, migration, and extracellular matrix remodeling. Accordingly, we have developed a collagen-based scaffold modified with combination of vancomycin-loaded liposomes and platelet derived growth factor (PDGF)-loaded DNA polyplexes. Both the liposomes and polyplexes were anchored to collagen using collagen mimetic peptides (CMPs). Our aim was to use CMP tethering to control the sequential release of vancomycin and PDGF polyplexes to immediately suppress infection and subsequently transfect wound bed fibroblasts with PDGF to assist the wound healing process. Vancomycin-loaded liposomes were prepared using dipalmitoylphosphatidylcholine (DPPC), cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG). The liposomes were 160.7±2.1 nm in diameter based on dynamic light scattering (DLS) analyses, and the loading capacity of vancomycin was 51.5±0.7% in the liposomes. PDGF polyplexes (115.2±1.2 nm in diameter) were prepared by self-assembly of polyethyleneimine and PDGF plasmid DNA (N/P = 8) in 20 mM HEPES buffer (pH = 6.0), and successful PDGF gene loading was confirmed by agarose gel electrophoresis. Co-gels were prepared with collagen (4 mg/mL), fibrinogen (1.25 mg/mL), and thrombin (0.156 IU/mL) combinations that could successfully encapsulate both the vancomycin-loaded liposomes and PDGF polyplexes. Drug release studies confirmed that ~80% of the vancomycin was released during the 48 h study period, whereas PDGF polyplexes were retained longer (\u3e 5 days) in the gel because their release requires collagen degradation mediated by matrix metalloproteinases present in the wound bed. The ability of the PDGF polyplexes to transfect fibroblasts was confirmed by in vitro cell transfection studies using green fluorescent protein (GFP) as a model gene. Furthermore, polyplex-mediated PDGF transfection was evaluated in fibroblasts cultured in an in vitro culture wound model, which showed that PDGF transfection enhanced migration rates of fibroblasts by ~2.4 fold as compared to controls in which culture wounds were allowed to heal in the absence of polyplexes. These results showcase the capacity for sequential delivery of vancomycin and PDGF gene in vitro, using collagen-based scaffolds, for potential applications in in vivo chronic wound treatments

Cell-mediated Delivery and Targeted Erosion of Noncovalently Crosslinked Hydrogels

A method for targeted delivery of therapeutic compounds from hydrogels is presented. The method involves administering to a cell a hydrogel in which a therapeutic compound is noncovalently bound to heparin

Covalent co-assembly between resilin-like polypeptide and peptide amphiphile into hydrogels with controlled nanostructure and improved mechanical properties

Covalent co-assembly holds great promise for the fabrication of hydrogels with controllable nanostructure, versatile chemical composition, and enhanced mechanical properties given its relative simplicity, high efficiency, and bond stability. This report describes our approach to designing functional multicomponent hydrogels based on photo-induced chemical interactions between an acrylamide-functionalized resilin-like polypeptide (RLP) and a peptide amphiphile (PA). Circular dichroism (CD) spectroscopy, electron microscopy, and amplitude sweep rheology were used to demonstrate that the co-assembled hydrogel systems acquired distinct structural conformations, tunable nanostructures, and enhanced elasticity in a PA concentration-dependent manner. We envisage the use of these materials in numerous biomedical applications such as controlled drug release systems, microfluidic devices, and scaffolds for tissue engineering

Biosynthetic Routes to Novel Macromolecular Materials

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