A Novel Nano-Ghosts Based System for Targeted Delivery of Antimir-221 and Stimulation of Cartilage Repair

Osteoarthritis is a leading cause of joint disability, with no disease-modifying treatments. Recently, RNA interference therapy has shown therapeutic potential, but there are no safe delivery vectors available. Our lab has developed an innovative drug delivery platform, termed Nano-Ghosts(NG), consisting in nano-vesicles reconstructed from the plasma membrane of mesenchymal stem cells’(MSCs). NG retain MSCs’ surface features and capacity for targeting inflammation sites. MicroRNA(miR)-221 inhibits chondrogenesis of MSCs, and its silencing leads to cartilage repair in vivo. Our aim was to develop a novel NG-antimiR-221 system for cartilage regeneration.

Electroporation was optimized for the encapsulation of fluorescently labelled antimiR-221. targeting of MSCs by antimiR-loaded NG was assessed using confocal microscopy and imaging flow cytometry analysis. NG endocytosis was studied by High Content Analysis(HCS). miR-221 silencing in MSCs was investigated by qPCR after incubating NG with MSCs cultured in monolayer(24h, 72h) or in a 3D Fibrin/hyaluronan hydrogel system(7d).

Our data showed 45% encapsulation efficiency of antimiR-221 in NG. Targeting of MSCs was achieved within 30 minutes. Imaging flow cytometry displayed high internalization of NG(>90%) and delivery of antimiR-221(>80%). Moreover, HCS kinetic studies indicated NG endosomal escape with less than 5% co-localization. Finally, strong silencing(>85%) of miR-221 in MSCs was achieved both under monolayer and 3D hydrogel culture condition, in a dose-dependent manner.

We developed an effective NG-antimiR-221 system exhibiting high MSC targeting ability and inducing miR-221 knockdown. Ongoing studies are assessing cartilage regeneration in vivo using an osteochondral defect mouse model. treated with HA-fibrin hydrogel containing NG-antimiR-221.