Rheological Characterization of Polysaccharide−Poly(ethylene glycol) Star Copolymer Hydrogels

Abstract

Binding interactions between low molecular weight heparin (LMWH) and heparin-binding peptides (HBP) have been applied as a strategy for the assembly of hydrogels that are capable of sequestering growth factors and delivering them in a controlled manner. In this work, the assembly of four-arm star poly(ethylene glycol) (PEG)-LMWH conjugate with PEG-HBP conjugates has been investigated. The interactions between LMWH and the heparin-binding regions of antithrombin III (ATIII) or the heparin interacting protein (HIP) have been characterized via heparin affinity chromatography and surface plasmon resonance (SPR); results indicate that the two peptides have slightly different affinities for heparin and LMWH, and bind LMWH with micromolar affinity. Solutions of the PEG-LMWH and of mixtures of the PEG-LMWH and PEG-HBP were characterized via both bulk rheology and laser tweezer microrheology. Interestingly, solutions of PEG-LMWH (2.5 wt % in PBS) form hydrogels in the absence of PEG-ATIII or PEG-HIP, with storage moduli, determined via bulk rheological measurements, in excess of the loss moduli over frequencies of 0.1-100 Hz. The addition of PEG-ATIII or PEG-HIP increases the moduli in direct proportion to the number of cross-links introduced. Characterization of the hydrogels via microrheology shows the gel microstructure is composed of polymer-rich fibrillar structures surrounded by polymer-depleted buffer. Potential applications of these hydrogels are discussed.

Manipulation of hydrogel assembly and growth factor delivery via the use of peptide–polysaccharide interactions

“…Our previous investigations of PEG-LMWH/PEG-HBP hydrogels suggested that the viscoelastic properties of the noncovalently assembled hydrogels were consistent with the heparin-binding kinetics of the peptides (from antithrombin III (ATIII) or heparin interacting protein (HIP)) [11]. Heparin-sepharose affinity chromatography and SPR experiments were therefore used to compare the heparin-binding affinity of PF4 ZIP with those measured for the previously prepared peptides [11] in order to confirm the potential of PF4 ZIP to mediate hydrogel formation.…”

“…Accordingly, heparin has been incorporated into covalent hydrogel delivery systems because of this ability to bind a diverse set of proteins. In addition, we and others have demonstrated that the interactions between heparin and specific HBPs can also mediate the assembly of noncovalently associated hydrogel networks [10][11][12].…”

“…The PF4 ZIP sequence CGGRMKQLEDKVKKLLKKNYHLENEVARLKKLVG is based on a GCN4 coiled-coil and mimics the heparin-binding domain of human platelet factor 4 [34]. The previously reported heparin-binding peptides, ATIII and HIP peptides, from antithrombin III (ATIII) and heparin interacting protein (HIP), included here for comparison, were synthesized according to similar protocols [10,11]. The crude peptides were purified via preparative-scale HPLC (Waters Delta 600, Waters Corporation, Milford, MA) equipped with a Waters Symmetry 300 C18 column (5 μm particle size, 19×150 mm).…”

“…Surface Plasmon Resonance (SPR). SPR sensorgrams were recorded on a BiaCore 3000 instrument (Biacore Inc, Piscataway, NJ), employing a high-capacity LMWH surface created on a commercially available streptavidin-coated chip via treatment with biotinylated LMWH, as previously described [11].…”

“…Heparin-sepharose affinity chromatography and SPR experiments were therefore used to compare the heparin-binding affinity of PF4 ZIP with those measured for the previously prepared peptides [11] in order to confirm the potential of PF4 ZIP to mediate hydrogel formation. Results of these experiments are summarized in Table 1.…”