ATP-activated decrosslinking and charge-reversal vectors for siRNA delivery and cancer therapy

Abstract

Stimuli-responsive polycations have been developed for improved nucleic acid transfection and enhanced therapeutic efficacy. The most reported mechanisms for controlled release of siRNA are based on polyelectrolyte exchange reactions in the cytoplasm and the degradation of polycations initiated by specific triggers. However, the degradation strategy has not always been sufficient due to unsatisfactory kinetics and binding of cationic fragments to siRNA, which limits the gene silencing effect. In this study, a new strategy that combines degradation and charge reversal is proposed.Methods: We prepared a polycation (CrossPPA) by crosslinking of phenylboronic acid (PBA)-grafted 1.8k PEI with alginate. It was compared with 25k PEI, 1.8k PEI and 1.8k PEI-PBA on siRNA encapsulation, ATP-responsive behavior and mechanism, cytotoxicity, cell uptake, siRNA transfection, in vivo biodistribution and in vivo anti-tumor efficacy. The in vitro and in vivo experiments were performed on 4T1 murine breast cancer cells and 4T1 tumor model separately.Results: The crosslinking strategy obviously improve the siRNA loading ability of 1.8k PEI. We validated that intracellular levels of ATP could trigger CrossPPA disassembly and charge reversal, which resulted in efficient and rapid siRNA release due to electrostatic repulsion. Besides, CrossPPA/siRNA showed strong cell uptake in 4T1 cells compared with 1.8k PEI/siRNA. Notably, the cytotoxicity of CrossPPA was pretty low, which was owing to its biodegradability. Furthermore, the crosslinked polyplexes significantly enhanced siRNA transfection and improved tumor accumulation. The high gene silencing ability of CrossPPA polyplex led to strong anti-tumor efficacy when using Bcl2-targeted siRNA.Conclusion: These results indicated that the ATP-triggered disassembly and charge reversal strategy provided a new way for developing stimuli-responsive siRNA carriers and showed potential for nucleic acid delivery in the treatment of cancer.

Interaction Kinetics of Peptide Lipids-Mediated Gene Delivery

“…A major intracellular barrier for DNA delivery is endosomal entrapment followed by tra cking and lysosomal degradation or exocytosis [37,38]. The escape of DNA from late endosomes/lysosomes into the cytosol is thought to be a rate-limiting step for many delivery approaches [39].…”

Virus‐inspired and mimetic designs in non‐viral gene delivery

“…Zhou et al . designed ATP‐responsive cross‐linked polycations (CrossPPA) to realize the burst release of siRNA at a high level of intracellular ATP, which simultaneously reduced the toxicity of delivery system and improved gene transfection.…”

“…53 Among them, systems based on PBA that can form a reversible ester linkage with cis-diol molecule are mainly reported. 54 Zhou et al 55 designed ATP-responsive cross-linked polycations (CrossPPA) to realize the burst release of siRNA at a high level of intracellular ATP, which simultaneously reduced the toxicity of delivery system and improved gene transfection. The polycation was prepared by cross-linking of PBA-grafted 1.8k PEI with alginate (as the diol linker), 55 which was initially strongly positively charged and could effectively pack siRNA.…”

Interaction Kinetics of Peptide Lipids-Mediated Gene Delivery

“…A major intracellular barrier for DNA delivery is endosomal entrapment followed by trafficking and lysosomal degradation or exocytosis [34,35]. The escape of DNA from endosomes into the cytosol is thought to be a rate-limiting step for many delivery approaches [36].…”