(B-64) Development of a Multivalent BMP-2 Presentation System from Glass Surfaces

Introduction:: Bone morphogenic proteins (BMP) are members of the TGF-beta superfamily and are responsible for many of the vital cellular responses in the human body. Specifically, BMP-2 is found to play an integral role in the growth and regeneration of cortical bone tissue and is one of the ligands responsible for the differentiation of osteocytes into osteoblasts. As such, BMP-2 is widely studied and has found use in therapeutics to aid bone regeneration [1]. This study aims to capture the potency of BMP-2 using a derivative amino acid sequence a fraction of the size of the BMP-2 protein. To achieve this, we have devised a method to present BMP-2 peptide from a glass surface in a multivalent fashion. The amino acid sequence used has been shown to induce osteogenesis [2] and this presentation method is made possible by utilizing strain-promoted alkyne-azide cycloaddition (SPAAC).


Materials and Methods:: Materials: Polymer backbones used consisted of hydroxyethyl acrylate (HEA) and NHS ester acrylate monomers. 5mm diameter glass cover slips were used, and surface functionalized with 3-azidopropyl silane. The polymer backbone was polymerized using a cyclopropenone-masked dibenzoazacyclooctyne (cp-DIBAC) [3] functionalized chain transfer agent. Monovalent presentation was achieved using cp-DIBAC alkene which allowed for DIBAC functionalization of the glass surface.

Polymer Synthesis: Polymer synthesis was achieved using light-initiated PET-RAFT polymerization. This was performed in a 96-well plate with monomer (95% HEA, 5% NHS acrylate, 1 M), CTA-DIBAC (5mM) and ZnTPP (2 mM) prepared in DMSO. Azide-functionalized BMP peptide was conjugated to the polymer backbone via copper-free DBCO-azide click chemistry.

Glass Functionalization: Cleaned and degreased cover slips were prepared prior to functionalization. The cover slips were etched using Piranha solution prior to being washed with water and dried in the desiccator. The glass surface was azide-functionalized by submerging cover slips in a 2% v/v silane in anhydrous toluene. Functionalized polymer chains were first put under a UV lamp for DIBAC deprotection before conjugated to the glass surface via SPAAC. To achieve monovalent peptide presentation, cp-DIBAC alkene was added to the glass surface via a copper catalyzed alkyne-azide click reaction.


Results, Conclusions, and Discussions:: To validate both monovalent and multivalent presentation, a fluorescent probe was used in place of BMP peptide. Monovalent presentation was validated using a Cy-5 azide dye (Fig 1. a,b) conjugated to deprotected cp-DIBAC on the glass surface. Multivalent presentation was validated using a 488-azide dye conjugated on the polymer backbone before being attached to the azide functionalized glass surface (Fig 1. c,d). As shown from the results, we can achieve ligand attachment onto the glass surface and the further validation of the deprotecting capabilities of cp-DIBAC will allow for more possibilities in terms of surface presentation design in future iterations.

This study validated the design method in successfully presenting BMP-2 peptide from a surface. Future studies will focus on developing a robust in vitro assay to measure the relative bioactivity of multivalent BMP peptides with varying backbone designs. Moreover, we seek to explore the capabilities of cp-DIBAC and its ability to be deprotected under UV light to further tune the design of the multivalent system.


Acknowledgements (Optional): : The authors acknowledge NIH NIGMS R35GM138296

References (Optional): : [1] El Bialy et al. Pharmaceutical Research, 34(6), 1152–1170. [2] Saito et al. Biochim Biophys Acta. 1651(1-2):60-7 [3] Li et al. J. Am. Chem. Soc. 2019 141 (50), 19823-19830