Background: Previously, we have demonstrated highly potent knockdown of POMC mRNA and significant reduction in secreted ACTH using siRNA, both in vitro and in vivo. Highly efficient delivery of siRNA to a target tissue is of critical importance for any therapeutic approach. Here we assess the suitability of a pH-sensitive polymer, PMPC-PDPA polymersomes encapsulating siRNA, as a novel delivery system as these have potential for tissue-directed delivery by co-complexing with appropriate antibodies, with intracellular release dependent on pH change after uptake.
Methods: The lowest effective dose of siRNA targeting POMC was assessed in vitro in AtT20 cells using a commercial lipid delivery agent. PMPC-PDPA polymersomes were devised and optimized used to encapsulate the siRNA and the physical characteristics determined. Immunofluorescence was used to investigate the presence of corticotrophin releasing hormone receptor 1 (CRHR1) on the surface of AtT20 cells.
Results: Suppression of POMC mRNA was demonstrated in a dose dependent manner. A 1 nM concentration of siRNA achieved a 50% reduction in ACTH compared to a negative siRNA control (P<0.001). Electron microscopy revealed PMPC-PDPA polymersomes encapsulating siRNA had a diameter of 100 nm, whilst having a neutral surface charge consistent with these particles having characteristics suitable for in vivo delivery. Real time quantitative PCR demonstrated the expression of CRHR1 mRNA in AtT20 cells and immunofluorescence demonstrated the presence of CRHR1 on the surface of AtT20 cells.
Conclusion: Polymersome-encapsulated siRNA targeting POMC have potential as a therapeutic for Cushings disease, with corticotroph targeting possible by antibody-particle complexes.