(489ag) Synthesis and Anticoagulant Function of Heparin Containing Block Copolymers On Polystyrene Microspheres

Heparinized coatings are commonly used in biomaterial surfaces that are in direct contact with blood. A number of case studies suggest that devices with grafted heparin show a diminished thrombogenic response, but results have not been uniformly favorable with respect to retained anticoagulant function. Suboptimal performance could be due in part to compromised heparin mobility and solvent accessibility upon immobilization. By taking into account the highly heterogeneous chemical composition of heparin and controlling the modification and binding chemistry, it might be possible to manufacture heparinized devices that retain a higher degree of anticoagulant activity. We set out to synthesize heparin-containing copolymer constructs, and to immobilize them at interfaces in a manner preserving heparin molecular mobility and solvent accessibility. For this purpose, internal thiol groups were introduced to unfractionated heparin (UFH) by reaction of carboxyl groups in its iduronic residues with 3, 3'dithiobis (propanoic) (DTP) hydrazide. Retention of anticoagulant activity after thiolation was tested by a chromogenic, anti-Factor Xa (anti-FXa) assay as well as the activated partial thromboplastin time (APTT). We determined, however, that only a small fraction of the original UFH activity was retained upon modification with DTP. The thiolated heparin derivatives were then linked to pyridyl disulfide (PDS)-activated poly[ethylene oxide]-poly[propylene oxide]-poly[ethylene oxide] (PEO-PPO-PEO) triblock copolymers, that had previously been bound to polystyrene microspheres (1.15 µm diameter) via hydrophobic association between the PPO block and polystyrene surface. Successful surface attachment of heparin was confirmed by monitoring the heparin linking reaction spectrophotometrically, and by zeta potential measurements recorded for uncoated, triblock-coated, and heparin-containing microsphere suspensions. Surface attachment of heparin in this manner was not observed to increase the anticoagulant activity of microsphere suspensions as determined either by APTT, or by the anti-FXa assay. This was likely due to the heparin having been modified on carboxylates in a random fashion (i.e., modified at multiple sites along the length of the heparin molecule as opposed to only at one end). We hypothesized that improved solvent accessibility of the pentasaccharide domain essential for heparin function, gained by modification of heparin only at a single site (its reducing end), would likely be essential for optimal function. Thiol groups were then introduced to UFH using an alternative chemistry to promote end-thiolation. In this case, an end-aminated form of UFH (HepNH2), was reacted with 2-iminothiolane (2-IT) under conditions favoring production of thiol groups at terminal primary amines over internal amines. Thiolation was quantified using Ellman's assay and o-phthalaldehyde. Anticoagulant activity after end-thiolation, determined by the anti-FXa and APTT assays, showed that HepNH2 modified by 2-IT retained the majority of original HepNH2 activity. Surface immobilized, PDS-activated triblocks were then used as above to attach end-thiolated heparin to polystyrene microspheres. Spectroscopic monitoring of the reaction indicated that quantities of surface immobilized heparin were not as large as those achieved using the original DTP coupling chemistry. And, the APTT assay showed no anticoagulant activity on heparinized microspheres, presumably due either to the presence of an insufficient amount of immobilized heparin, or to steric constraints inhibiting the formation of a functional heparin-antithrombin complex. However, immobilized heparin did retain good anti-FXa activity, with significantly greater activity being recorded at surfaces treated with thiolated HepNH2 (attached end-on) relative to those treated with thiolated UFH (attached side-on, by reaction with internal amines).