Plasmin-Cleavable Nanoparticles for On-Demand Release of Morphogens in Vascularized Osteogenesis

The objective of this work was to engineer self-assembled nanoparticles (NPs) for on-demand release of bone morphogenetic protein-2 (BMP2) and vascular endothelial growth factor (VEGF) in response to enzymes secreted by the migrating human mesenchymal stem cells (hMSCs) and human endothelial colony forming cells (ECFCs) to induce osteogenesis and vasculogenesis. Gene expression profiling experiments revealed that hMSCs and ECFCs, encapsulated in osteogenic/vasculogenic hydrogels, expressed considerable levels of plasminogen, urokinase plasminogen activator and its receptor uPAR, and tissue plasminogen activator. Therefore, the plasmin-cleavable lysine-phenylalanine-lysine-threonine (KFKT) was used to generate enzymatically cleavable NPs. The acetyl-terminated, self-assembling peptide glycine(phenylalanine)(3)GFFF-ac and the plasmin-cleavable GGKFKTGG were reacted with the cysteine-terminated CGGK(Fmoc/MTT) peptide through the MTT and Fmoc termini, respectively. The difunctional peptide was conjugated to polyethylene glycol diacrylate (PEGDA) with molecular weights (MW) ranging from 0.5 to 7.5 kDa, and the chain ends of the PEG-peptide conjugate were terminated with succinimide groups. After self-assembly in aqueous solution, BMP2 was grafted to the self-assembled, plasmin-cleavable PEG-based (PxSPCP) NPs for on-demand release. The NPs' stability in aqueous solution and that of the grafted BMP2 were strongly dependent on PEG MW. P2SPCP NPs showed high particle size stability, BMP2 grafting efficiency, grafted protein stability, and high extent of osteogenic differentiation of hMSCs. The localized and on-demand release of BMP2 from PxSPCP NPs coencapsulated with hMSCs in the linear polyethylene glycol-co-lactide acrylate patterned hydrogel with microchannels encapsulating hMSCs + ECFCs and VEGF-conjugated nanogels resulted in the highest extent of osteogenic and vasculogenic differentiation of the encapsulated cells compared to directly added BMP2/VEGF. The on-demand release of BMP2 from PxSPCP NPs not only enhances osteogenesis and vasculogenesis but also potentially reduces many undesired side effects of BMP2 therapy in bone regeneration.