A novel nano-ghosts based system for targeted delivery of antimir-221 and stimulation of cartilage repair

Osteoarthritis (OA) is a leading cause of joint disability worldwide, characterized by articular cartilage degradation, with no disease-modifying treatments available. In recent years, RNA interference (RNAi) therapy has shown great potential, but the clinical translation is hindered by the lack of safe and effective delivery systems. Recently, our lab has developed an innovative drug delivery platform, termed Nano-Ghosts (NGs), consisting of nano-vesicles reconstructed from the plasma membrane of mesenchymal stem cells (MSCs). The NGs retain MSCs’ surface features and targeting capabilities towards tumor and inflammation sites¹. Moreover, our group demonstrated the efficacy of NGs delivery system for gene therapy². MicroRNA(miR)-221 is a negative regulator of chondrogenesis, and its silencing in MSCs leads to chondrogenic differentiation and cartilage repair in vivo³. Our aim was to develop a novel NGs-based delivery system of antimiR-221 for targeted and efficient miRNA silencing in MSCs.

Our data showed that antimiR-221 can be efficiently loaded in the NGs using electroporation, achieving high encapsulation efficiency (up to 45%), as confirmed by fluorescent quantification and cryo-TEM imaging. Confocal microscopy and flow cytometry analyses demonstrated targeting of NGs towards MSCs within 30 minutes. MSCs viability was not affected by the treatment, as confirmed by Alamar Blue assay. Imaging flow cytometry showed MSCs were efficiently transfected with antimiR-221-NGs within 30 minutes, with a cellular internalization of NGs >90%, and a cytosolic presence of antimiR-221 >80%. Moreover, high-content screening kinetic studies showed that NGs underwent only minimal endo-lysosomal recycling, with less than 5% and 9% co-localization, respectively. Finally, qRT-PCR analysis revealed a strong silencing of miR-221 at 24 and 72 hours (75% and 83%, respectively) after treatment of MSCs with 5 µg/mL antimiR-221-loaded NGs.

In conclusion, we developed a novel and effective NGs-based system for RNAi molecules. AntimiR-221-loaded NGs were shown to efficiently transfect MSCs, leading to rapid and strong silencing of the anti-chondrogenic miR-221. Our delivery system may be employed for targeting endogenous MSCs in a context of trauma or OA, thus stimulating and improving cartilage repair.

1. Furman T, et al., Nano Letters, 2013
2. Kaneti L, et al., Nano Letters, 2016.
3. Lolli A, et al., Stem Cells, 2016