Peptide amphiphiles (PAs) are self-assembling molecules that form supramolecular nanostructures of different shapes and have shown great potential as biomaterials with applications in regenerative medicine. However, the effects of PA nanostructure morphology on growth factor delivery for cell differentiation have not yet been examined. We have previously reported on a PA nanostructure that was designed to display a sequence that binds to bone morphogenetic protein 2 (BMP2). BMP2 is a critical growth factor that stimulates osteogenesis, and the BMP2-binding PA (BMP2b) was found to promote bone regeneration in a spinal fusion model with much lower dosage of BMP2 than standard treatments. Interestingly, <i>in vitro</i> work showed that the BMP2b PA is most effective when co-assembled with a backbone PA (E3) without a bioactive group. Since it is known that endocytosis of growth factors reduces their effectiveness, we hypothesized that complexation of the BMP2 protein with the nanostructures of the co-assembled PA molecules reduces the extent of internalization compared to the pure BMP2b PA. We present here results of transmission electron microscopy and atomic force microscopy demonstrating that E3-BMP2b co-assembly forms long fiber bundles, while pure E3 PA forms long fibers and pure BMP2b PA fibers forms short fibers. We then investigated the relationship between PA nanostructure morphology and the rate of cellular internalization to explain the BMP2 protein binding and release behavior of the co-assembly. Using flow cytometry and confocal microscopy, we show that the co-assembly exhibits slower PA uptake by C2C12 pre-myoblast cells known to model osteoblast differentiation, compared to E3 or BMP2b PA by themselves. We also demonstrate that the large bundles of the co-assembly increase protein binding and stabilization by PAs, protecting BMP2 from cellular internalization and degradation. We conclude that varying the size and morphology of nanofibers can help shield proteins from cellular uptake and prolong the effect of therapeutic protein in bone regeneration.

Effects of supramolecular nanostructure morphology on protein delivery

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