Pullulan-based polymeric nanosystem for KEAP1 siRNA delivery: Mitigating ROS and promoting wound healing in vitro

Recently, siRNA delivery has shown considerable potential for therapeutic use. While commercial options facilitate efficient delivery, they can be toxic to cells to a certain degree. Therefore, using relatively less toxic and safe delivery vehicles can increase the efficiency of therapy. This study examines the utilization of Pullulan-poly(ethyleneimine) nanoparticles (Pullulan-PEI-NPs) as an effective non-viral delivery system for Kelch-like ECH-associated protein 1 (KEAP1) siRNA, with the aims of mitigating oxidative stress and facilitating wound healing in HaCaT cells by enhancing cellular antioxidant mechanisms. The Pullulan-PEI-NPs were synthesized and characterized with Dynamic Light Scattering, Nanoparticle Tracking Analysis, Fourier Transform Infrared Spectroscopy, and electron microscopy techniques. The polyplexes were formed via electrostatic interaction between Pullulan-PEI-NPs and KEAP1 siRNA, and successful siRNA delivery was confirmed by cellular uptake analysis, mRNA expression studies, and KEAP1 and nuclear factor E2-related factor 2 (NRF2) protein level assessments. Pullulan-PEI-NPs had a silencing efficiency of 72 %, which is very close to the commercial transfection agent Lipofectamine 3000 (80 %). In following studies, KEAP1 siRNA delivery with Pullulan-PEI-NPs mitigates TBHP and H₂O₂ mediated ROS and facilitates wound healing with 98.9 % wound closure in a scratch assay on HaCaT cells. Obtained results highlight the therapeutic relevance of KEAP1 silencing in redox-regulated wound regeneration. This is the first report showing that Pullulan-PEI based cationic polymeric nanoparticles can be used to deliver KEAP1 siRNA and evaluate its role in mitigating ROS and increasing wound healing. These results highlight the promise of Pullulan-PEI-NPs as a safe and adaptable platform for non-viral gene therapy applications.