(516g) Award Submission: Conjugation of rhBMP-2 Derived Peptide to Self-Assembled Nanoparticles Enhances Osteogenic Differentiation of Mesenchymal Stem Cells
AIChE Annual Meeting
Wednesday, November 10, 2010 - 5:08pm to 5:26pm
Introduction Short half-life, diffusion of the protein away from the site of interest, and bone overgrowth limit the use of bone morphogenetic proteins in clinical applications [1,2]. The peptide sequence KIPKA SSVPT ELSAI STLYL, corresponding to residues 73-92 of the rhBMP-2 protein, has shown affinity to the BMP receptors and can induce osteogenesis . We propose grafting this peptide to our biodegradable poly(lactide fumarate) (PLAF), poly(lactide-co-glycolide fumarate) (PLGF), and poly(lactide ethylene oxide fumarate) (PLEOF) nanoparticles (NPs) to deliver it in a sustained manner while providing a high surface density of the peptide for interaction with the receptors of the BMP. The objective of this work was to measure the release of the grafted peptide from these NPs and determine the response of the bone marrow mesenchymal stem (BMS) cells cultured with the conjugated NPs with respect to osteogenic and vasculogenic activity based on calcium deposition, cell numbers, and expression levels of alkaline phosphatase (ALP), osteocalcin (OC), osteopontin (OP), and Pecam1.
Methods PLAF, PLEOF, and PLGF were synthesized and functionalized with fumaryl chloride as described . The peptide sequence KIPKA SSVPT ELSAI STLYL was synthesized in solid-phase by Fmoc chemistry and functionalized with a cysteine residue . NPs were self-assembled by dissolving 45 mg PLAF (or PLGF) and 5 mg PLEOF in organic solvent and dialysis of the solution against PBS. 10 mg of peptide were conjugated by reaction of the cysteine residue to the fumarate group of the macromers. After dialysis, the suspension was centrifuged to precipitate the particles and remove unreacted peptide. The NPs were then resuspended in PBS. Release was determined my measuring the amount of peptide in solution every 24 hours for 28 days. Measurements were done by using a Kaiser reagent to determine the amount of amines in solution and correlated to the total amount of peptide grafted on their surface. For osteogenic activity measurements, NPs were grafted with 200 ng/mL BMP peptide and suspended in cell culture media after conjugation. BMS Cells were seeded at a density of 5×104 cells/cm3 and exposed to free rhBMP-2 protein, PLAF and PLGF NPs directly, and their supernatants for 21 days. After days 4, 7, 14, and 21, cells were lysed and analyzed for DNA content, ALP activity, Ca concentration, and expression level of OC, OP and Pecam1.
Results Grafting efficiency was relatively high at 69.1±4.6% for PLAF/PLEOF and 85.5±2.3% for PLGF/PLEOF NPs. Release of BMP peptide from PLGF-NHS NPs was linear for 26 days. PLAF-NHS NPs displayed a similar trend, with slower linear release until 32 days. Release could account for all of the peptide grafted to the surface. DNA content decreased for osteogenic groups as cells differentiated and underwent mineralization. ALP activity peaked at 14 days for osteogenic groups. Calcification was evident after this time for all groups, indicating osteogenic potential of grafted peptide on the NPs. A significant difference, however, was seen for OP, OC and Pecam1 expression, with 4-5 times higher OC and Pecam1 expression and 2 times higher for OP for BMS cells cultured with rhBMP-2 peptide grafted NPs.
Discussion The sustained release of the BMP peptide from the NPs with high BMP peptide surface density provided a higher expression of OP and OC, as compared with the free BMP. The higher Pecam1 activity could be related to the higher expression of OP and provides evidence for the vasculogenic potential of the BMP peptide grafted NPs. The sustained release of BMP peptide from the NPs can potentially provide an efficient way for inducing osteogenesis and bone formation in clinical applications.
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