Preparation and Evaluation of Polymeric Hybrid Micelles to Co-Deliver Small Molecule Drug and siRNA for Rheumatoid Arthritis Therapy

Small interfering RNA (siRNA) has emerged as a potential drug candidate in the treatment of various diseases. However, efficient delivery of siRNA molecules to the cytoplasm of target cells still remains a huge challenge for the application in vivo. Here, this protocol describes a practical approach to prepare a highly flexible polymeric hybrid micelles for the in vivo delivery of siRNA. The micelles consist of cationic polycaprolactone-polyethylenimine (PCL-PEI) and neutral polycaprolactone-polyethylene glycol (PCL-PEG), the ratio of which could be rationally adjusted to obtain various hybrid micelles with different physicochemical properties. The small proportion of PEI segments ensures the sufficient encapsulation of siRNA without causing obvious cytotoxicity. The high proportion of PEG segments renders the hybrid micelles nearly neutral surface charge and prolonged in vivo circulation. In order to efficiently block the key inflammatory signaling NF-κB pathway in rheumatoid arthritis (RA), siRNA targeting p65 (a key subunit of NF-κB family) combined with small molecule drugs dexamethasone (Dex) using the hybrid micelle as a carrier has been developed for the treatment of RA. These hybrid micelles would encapsulate siRNA via the electrostatic interaction between siRNA and PEI and load Dex in the PCL core via the hydrophobic interaction. The dual drugs are expected to be delivered to inflamed sites to synergistically suppress the NF-κB signaling and achieve the improved anti-inflammatory efficacy. Here, the methods for preparation and characterization of the hybrid micelles are described in detail. And the in vitro evaluation including the endosome escape and gene silencing have been systematically indicated. Meanwhile, the in vivo biodistribution and pharmacodynamics of this dual drugs-loaded formulation have been assessed according to the normalized methods. This protocol displays a practical approach for co-delivering genetic drugs and small molecule chemotherapeutics for in vivo application.