Controlling mesenchymal stem cell (MSC) differentiation by RNA interference (RNAi) is a promising approach for next-generation regenerative medicine. However, efficient delivery of RNAi therapeutics is still a limiting factor. In this study, we have developed a simple, biocompatible, and highly effective delivery method of small RNA therapeutics into human MSCs (hMSCs) from an implant surface by calcium ions. First, we demonstrated that simple Ca/siRNA targeting green fluorescent protein (GFP) nanocomplexes were able to efficiently silence GFP in GFP-expressing hMSCs with adequate Ca2+ concentration (>5 mM). In addition, a single transfection could obtain a long-lasting silencing effect for more than 2 weeks. All three of the main endocytosis pathways (clathrin- and caveolin-mediated endocytosis and macropinocytosis) were involved in the internalization of the Ca/siRNA complexes by MSCs, and macropinocytosis plays the most dominant role. Furthermore, the Ca/siRNA complexes could be efficiently loaded onto the titanium implant surface when pretreated with anodization to create a nanotube (NT) layer. Because of the hydrophilic property of the NT surface, the Ca/siRNA was quickly loaded (less than 4 h) with high efficiency (nearly 100%), forming an even amorphous coating. The Ca/siRNA-coated NT surface showed an initial burst release of 80% of the siRNA complexes over 2 h, which is adequate to achieve robust gene silencing of attached hMSCs. To demonstrate the therapeutic potential of our Ca/siRNA coating technology, Ca/antimiR-138 complexes were loaded on to the NT surface, which strongly enhanced the osteogenic differentiation of hMSCs. In conclusion, our findings suggest that Ca2+ is an effective and biocompatible carrier to deliver small RNA therapeutics into hMSCs, both in solution and from functionalized surfaces, which provides a novel approach to control the MSC differentiation and tissue regeneration.
Keywords: calcium; mesenchymal stem cells; microRNA; osteogenesis; siRNA; titanium implant.