Exploitation of high tumour GSH levels for targetted siRNA delivery in rhabdomyosarcoma cells
Description
Background: Metastatic alveolar rhabdomyosarcoma (aRMS) is an aggressive paediatric cancer with a poor prognosis. Downregulation of critical tumour genes using targetted siRNA has the potential to improve patient outcomes. However, the delivery of siRNA remains an obstacle, and association with nanoparticles could help to deliver, protect, target, and enhance penetration. The addition of near-infrared (NIR) dyes to the nanosystem can enable monitoring of uptake and accumulation during treatment. Methods: In this study, two siRNA targeting options were selected and validated: (i) Human αB-crystallin (CRYAB) & Heat Shock Protein Family B (Small) Member 2 (HSPB2), and (ii) Keratin 17 (KRT17). A mesoporous silica based theranostic nanosystem was loaded with IR820 dye and linked to the siRNA via disulfide bonds. The system was characterized and evaluated for transfection efficiency and signalling. In addition, the metabolic effects and cell proliferation were monitored in 2D culture and also in 3D spheroid models. Results: The synthesized nanosystem was found to maintain a strong bond with the siRNA molecules, and showed a high degree of protection; preventing degradation with RNase I during a 24 h incubation. The siRNA was effectively delivered and transfected into the aRMS cells by the nanosystem with a significant suppression of viability; 53.21 ± 23.40 % for CRYAB & HSPB2 siRNA, and 88.06 ± 17.28 % for KRT17 siRNA. The cause of cellular proliferation suppression was discovered with over 50% of cell apoptosis and necrosis within the RH30 spheroids were found after 72 h of nanosystem delivered siRNA transfection. Moreover, it has been demonstrated that the IR820 cellular uptake rate and penetration depth was significantly improved through the stable loading into the nanosystem. Conclusions: A successful theranostic nanosystem was generated that combined targeting siRNA treatment with IR820 NIR monitoring. This may help to pave the way for less invasive and more effective treatment of aRMS.