Dual-Functionalized Graphene Oxide Based siRNA Delivery System for Implant Surface Biomodification with Enhanced Osteogenesis

Surface functionalization by small interfering RNA (siRNA) is a novel strategy for improved implant osseointegration. A gene delivery system with safety and high transfection activity is a crucial factor for an siRNA-functionalized implant to exert its biological function. To this end, polyethylene glycol (PEG) and polyethylenimine (PEI) dual-functionalized graphene oxide (GO; nGO-PEG-PEI) may present a promising siRNA vector. In this study, nanosized nGO-PEG-PEI was prepared and optimized for siRNA delivery. Titania nanotubes (NTs) fabricated by anodic oxidation were biomodified with nGO-PEG-PEI/siRNA by cathodic electrodeposition, designated as NT-GPP/siRNA. NT-GPP/siRNA possessed benign cytocompatibility, as evaluated by cell adhesion and proliferation. Cellular uptake and knockdown efficiency of the NT-GPP/siRNA were assessed by MC3T3-E1 cells, which exhibited high siRNA delivery efficiency and sustained target gene silencing. Casein kinase-2 interacting protein-1 (Ckip-1) is a negative regulator of bone formation. siRNA-targeting Ckip-1 (siCkip-1) was introduced to the implant, and a series of in vitro and in vivo experiments were carried out to evaluate the osteogenic capacity of NT-GPP/siCkip-1. NT-GPP/siCkip-1 dramatically improved the in vitro osteogenic differentiation of MC3T3-E1 cells in terms of improved osteogenesis-related gene expression, and increased alkaline phosphatase (ALP) production, collagen secretion, and extracellular matrix (ECM) mineralization. Moreover, NT-GPP/siCkip-1 led to apparently enhanced in vivo osseointegration, as indicated by histological staining and EDX line scanning. Collectively, these findings suggest that NT-GPP/siRNA represents a practicable and promising approach for implant functionalization, showing clinical potential for dental and orthopedic applications.