(262d) Structure and Stability of Pol(L-lysine)/Hyaluronan Thin Films As Nanoreservoirs for the Bone Morphogenetic Protein-2

Polyelectrolyte multilayer films (PEM) made of poly(L-lysine) and hyaluronan (PLL/HA) have been recently investigated for their ability to trap the osteoinductive recombinant human bone morphogenetic protein-2 (rhBMP-2) (Crouzier et al., 2009). However, several tests have to be performed prior to their possible use in clinics as bioactive coatings on orthopedic or dental implants. First, all medical devices should sustain a sterilization process before in vivo implantation and they should also sustain storage for at least several months. In view of future applications of the PEM films, we first aimed here to investigate the stability, shelf life-time and sterilization of (PLL/HA) film. Secondly we aimed to investigate the secondary structure of rhBMP-2 trapped hydrated or dry (PLL/HA) films, as compared to the protein in solution at different pHs. To this end, we used Fourier Transform Infrared Spectroscopy (FTIR) to investigate the structure of (PLL/HA) films in dry state, their stability over time, the effects of sterilization by gamma irradiation and the structure of rhBMP-2 trapped in the films. We found that the (PLL/HA) films were very stable over the 2-month period, with less than 2% variation in the absorbance intensity of the amide I band. We also proved that the film can withstand the sterilization process without any major change in their structure. We quantified the secondary structure of rhBMP-2 trapped in a hydrated film and observed that it exhibited a similar structure to that of the protein in solution at pH 3 (representing the optimal conditions for BMP-2 loading the film). The preservation of the secondary structure of the film when trapped and confined in the PEM film may explain why rhBMP-2 remained bioactive. Interestingly, when the protein was trapped in dry films, a significant change in the protein structure was visible with the appearance of intermolecular b-sheets. We attributed these changes to protein/protein interactions or to protein/PLL interactions.

Crouzier, T., K. Ren, C. Nicolas, C. Roy, and C. Picart. 2009. Layer-by-Layer films as a biomimetic reservoir for rhBMP-2 delivery: controlled differentiation of myoblasts to osteoblasts. Small. 5:598-608.