Insights into siRNA Transfection in Suspension: Efficient Gene Silencing in Human Mesenchymal Stem Cells Encapsulated in Hyaluronic Acid Hydrogel

摘要

Small interfering RNAs (siRNAs) are powerful tools for post-transcriptional gene silencing, which offers enormous opportunities for tissue engineering applications. However, poor serum stability, inefficient intracellular delivery, and inevitable toxicity of transfection reagents are the key barriers for their clinical translation. Thus, innovative strategies that allow safe and efficient intracellular delivery of the nucleic acid drugs at the desired site is urgently needed for a smooth clinical translation of therapeutically appealing siRNA-based technology. In this regard, we have developed an innovative siRNA transfection protocol that employs a short incubation time of just 5 min. This allows easy transfection in suspension followed by transplantation of the cells in a hyaluronic acid (HA) hydrogel system. We also report here the unique ability of siRNA to bind HA that was quantified by siRNA release and rheological characterization of the HA-hydrogel. Such interactions also showed promising results to deliver functional siRNA in suspension transfection conditions within 30 min using native HA, although removal of excess HA by centrifugation seem to be essential. In the 2D experiments, suspension transfection of hMSCs with RNAiMAX resulted in approximate to 90% gene silencing (with or without removal of the excess reagent by centrifugation), while HA demonstrated a modest approximate to 40% gene silencing after removal of excess reagent after 30 min. Transplantation of such transfected cells in the HA-hydrogel system demonstrated an improved knockdown (approximate to 90% and approximate to 60% with RNAiMAX and HA respectively after 48 h), with lower cytotoxicity (up to 5-days) as determined by PrestoBlue assay. The gene silencing efficiency in the 2D and 3D conditions were also confirmed at the protein levels by Western blot analysis. We postulate this novel transfection method could be applied for in vivo applications as it allows minimal manipulation of cells that are to be transplanted and reduce toxicity.