Impact of cryoprotectants and formulation processes on the stability and efficacy of siRNA-loaded cationic bicelles.

The upregulation of vascular cell adhesion molecule-1 (VCAM-1) by the transcription factor GATA-6 during inflammation of the brain is a key contributor to neuronal injury. Silencing GATA-6 expression in brain endothelial cells may provide an avenue for ameliorating blood-brain barrier (BBB) inflammation and slowing neurodegeneration. Cationic bicelles composed of DPPC/DC7PC/DOTAP at a molar ratio of 63.8/25.0/11.2 were designed for the targeted delivery of GATA-6 siRNA to the BBB. However, formulation processes such as sonication or lyophilization introduce stresses that may impact the structural stability of the bicelles and therapeutic efficacy of the siRNA cargo. Therefore, these processes must be optimized. A sonication time of 3 min was sufficient for achieving a narrow particle size distribution without mechanically damaging the siRNA. A trehalose concentration of 10% w/v was effective at protecting the siRNA-bicelle complexes from particle aggregation and loss of silencing efficiency after 3 cycles of freeze-thaw. By contrast, a sucrose concentration of 5% w/v was only able to protect the siRNA-bicelle complexes from particle aggregation. Moreover, the addition of sucrose partially hindered the cellular uptake of cationic bicelles in polarized human cerebral microvascular endothelial cell (hCMEC/D3) monolayers, suggesting that it is an inappropriate cryoprotectant for cationic bicelles. Optimization of these formulation parameters for siRNA-bicelle complexes contributes towards their efficacy during delivery and stability during storage, which are critical factors to consider in a clinical setting. Thus, these findings will aid in the translation of siRNA-loaded cationic bicelles from the bench to the bedside.