In the present study, we report 2-nitrobenzenesulfonamide group as a chemical linker for construction of siRNA-polymer conjugates. Chemical linker with bioresponsibility (e.g., cleavability in the cell) is necessary for design of bio-responsive materials including siRNA-polymer conjugates, in order to realize site-specific functions. Among the biological stimuli, redox potential has been often utilized due to its huge difference across cellular membrane; however, the current redox-sensitive linker is limited mostly to disulfide group. In this regard, the moderate cleavage of disulfide group in extracellular milieu has led to increasing demand of another redox-sensitive chemical linker. To this end, we found that 2-nitrobenzenesulfonamide group has enhanced stability at extracellular reductive condition, compared to conventional disulfide group, and can be cleaved by combination of glutathione (GSH) and glutathione-<i>S</i>-transferase (GST), which resides mostly in the cell.<br/>PEG-siRNA conjugates containing disulfide linker (PEG-disulfide-siRNA) and carboxylic amide linker (PEG-car-siRNA), as well as 2-nitrobenzenesulfonamide linker (PEG-sul-siRNA), were prepared as controls of conventional system and non-cleavable system, respectively. An incubation in a mimicked extracellular condition (20 μM GSH) induced siRNA release from PEG-disulfide-siRNA (38%, 72 hours), whereas siRNA release was suppressed for PEG-sul-siRNA system (7%, 72 hours), suggesting the improved stability of the 2-nitrobenzenesulfonamide linker in extracellular environment. In contrast, an incubation in a mimicked intracellular condition (1 mM GSH) induced siRNA release from PEG-sul-siRNA (20%, 72 hours), where the combination of GSH and GST (1mM GSH and 0.019 mg/mL GST) accelerated siRNA release (90%, 72 hours incubation), indicating the pivotal role of combination of GSH and GST for efficient cleavage of 2-nitrobenzenesulfonamide group. The intracellular cleavage of 2-nitrobenzenesulfonamide group was confirmed by gene silencing assay for cultured cells; PEG-sul-siRNA induced higher gene silencing efficacy, compared to PEG-car-siRNA, suggesting intracellular cleavage of 2-nitrobenzenesulfonamide group for siRNA release and effective gene silencing induction.

2-Nitrobenzenesulfonamide group as a chemical linker with highly selective cleavability in response to intracellular redox system for a construction of siRNA-polymer conjugate

253rd National Meeting (2017)

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