Methods: Methods: Poly(lactide fumarate) PLAF, PLGF, and poly(lactide-co-ethylene oxide fumarate) (PLEOF) were synthesized by condensation polymerization. Succinimide-terminated macromers (PLAF-NHS or PLGF-NHS) were produced by reacting the hydroxyl end-groups of the PLAF or PLGF macromers with the carbonate group of N,N'-Disuccinimidyl carbonate (DSC). PLAF-NHS and PLGF-NHS NPs were produced by dialysis of the macromers in dimethylsulfoxide (DMSO)/ N, N-dimethylformamide (DMF) against water. The morphology and size distribution of the NPs was examined with ESEM. The size distribution of NPs was measured by dynamic light scattering. Degradation of the NPs was followed by measuring their mass as a function of incubation time. For conjugation, rhBMP-2 (1 µg/ml in PBS) was allowed to react with the succinimide terminated NPs under ambient conditions for 12 h. For determination of release kinetics, 1 mg rhBMP-2 loaded NPs were incubated with 1 ml PBS (pH 7.4) at 37°C with orbital shaking. At each time interval, the suspension was centrifuged at 15,000 rpm, and the supernatant was removed for analysis. The enzymatically active concentration of the BMP released from the microspheres was measured by enzyme-linked immunosorbent assay (ELISA) using the BMP Quantikine kit(R&D Systems).
Results: The average size of PLAF-NHS and PLGF-MHS NPs was 325 and 250 nm, respectively. The conjugation efficiency was determined by measuring the amount of free rhBMP-2 after the conjugation reaction. Conjugation efficiency was >95% for PLAF-NHS and PLGF-NHS NPs. The release of rhBMP-2 from PLAF-NHS NPs was linear with time and >0.4 µg was released after 2 weeks; the release from PLGF-NHS NPs was non-linear and >0.2 µg was released after 2 weeks. PLGF-NHS and PLAF-NHS NPs completely degraded in 25 and 38 days which demonstrated that the release was dominated by erosion of the matrix.
Conclusion: Since proteins can be immobilized on PLGF-NHS NPs by the reaction of succinimide groups with amine groups of the protein at physiological condition, conjugated NPs are potentially useful for immobilization and sustained release of differentiation factors like rhBMP-2 in tissue engineering applications.